Binding proteins and methods of use thereof

ABSTRACT

The present disclosure provides binding proteins, such as antibodies, that bind to a GDNF Family Receptor Alpha Like (GFRAL) protein, including human GFRAL protein, and methods of their use.

FIELD

This application is a divisional of U.S. application Ser. No.15/449,839, filed Mar. 3, 2017, which claims the benefit of U.S.Provisional Application No. 62/316,516, filed Mar. 31, 2016, the entirecontents of each of which are incorporated herein by reference.

The present disclosure relates generally to binding proteins, such asantibodies, that bind to a GDNF Family Receptor Alpha Like (GFRAL)protein, including human GFRAL protein, and methods of their use.

BACKGROUND

Growth differentiation factor 15 (GDF15) is a protein belonging to thetransforming growth factor beta (TGF-β) superfamily. GDF15 is also knownas TGF-PL, MIC-1, PDF, PLAB, NAG-1, and PTGFB. GDF15 mRNA is reported tobe most abundant in the liver, with lower levels seen in some othertissues. Its expression in liver can be significantly up-regulated ininjury of organs such as liver, kidney, heart and lung.

GDF15 is reported to play a role in regulating inflammatory andapoptotic pathways in injured tissues and during disease processes. Ithas been reported that GDF15 is a mediator of cachexia in variousdiseases. However, cachexia is a complex and incompletely understoodsyndrome. In addition, at least some tumors over-express and secreteGDF15, and elevated serum GDF15 levels have been associated with variouscancers. GDF-15 has been described as a negative regulator of macrophageactivation by suppressing the release of TNF-α, IL-1, IL-2 and MCS-F,thus inhibiting the positive feedback of local inflammatory signalingsimilar to the effects of TGF-β. Monoclonal antibodies against GDF15have been disclosed as potential therapeutic agents for the treatment ofcachexia and of cancer. The receptor for GDF15 is unknown.

There is a significant unmet need for therapeutic agents effective totreat weight loss associated with a number of diseases and conditions,including wasting diseases such as cachexia or sarcopenia andinflammatory conditions such as systemic inflammation or an acuteinflammatory response. There is also a significant unmet need fortherapeutic agents effective to treat chronic diseases, includingcancer, chronic renal disease, chronic obstructive pulmonary disease,AIDS, tuberculosis, chronic inflammatory disease, systemic inflammation,and muscle wasting diseases, including in which involuntary body weightloss and/or muscle mass loss is involved.

SUMMARY

The present disclosure provides proteins that bind to a GDNF FamilyReceptor Alpha Like (GFRAL) protein, including binding proteins such asantibodies that bind to a GFRAL protein. Such binding proteins includingantibodies, may bind to a GFRAL polypeptide, a GFRAL fragment and/or aGFRAL epitope. Such binding proteins, including antibodies, may beantagonists (e.g., inhibit binding of a GDF15 protein to a GFRALprotein, inhibit binding of a RET protein to a GFRAL protein, inhibit aGDF15 protein induced signaling, and/or inhibit formation of aGDF15/GFRAL or a GDF15/GFRAL/RET receptor complex).

The present disclosure also provides binding proteins, includingantibodies (e.g., monoclonal antibodies) or fragments thereof, that (i)bind to a GFRAL protein, (ii) inhibit binding of a GDF15 protein to aGFRAL protein, and/or (iii) inhibit binding of a RET protein to a GFRALprotein.

In some embodiments, the anti-GFRAL antibodies are humanized antibodiesthat bind to a GFRAL polypeptide, a GFRAL fragment, or a GFRAL epitope.In certain embodiments, an anti-GFRAL antibody comprises a VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibodydesignated 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5,2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6,P1H8, or P8G4 as described herein, or a humanized variant thereof. Incertain embodiments, an anti-GFRAL antibody can further comprise a VHFR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3, and/or VL FR4 of ahuman immunoglobulin amino acid sequence or a variant thereof.

In some embodiments, a binding protein (e.g., an anti-GFRAL antibody)comprises six CDRs or less than six CDRs. In some embodiments, a bindingprotein (e.g., an anti-GFRAL antibody) comprises one, two, three, four,five, or six CDRs selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and/or VL CDR3. In some embodiments, a binding protein (e.g., ananti-GFRAL antibody) comprises one, two, three, four, five, or six CDRsselected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VLCDR3 of a monoclonal antibody designated as 1C1, 3P10, 12A3, 5F12, 5A20,8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9,24G2, 6G9, 2611, 1A3, P1B6, P1H8, or P8G4 as described herein, or ahumanized variant thereof. In some embodiments, a binding protein (e.g.,an anti-GFRAL antibody) further comprises a scaffold region or frameworkregion, including a VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VLFR3, and/or VL FR4 of a human immunoglobulin amino acid sequence or avariant thereof.

In some embodiments, the antibody is a humanized antibody, a monoclonalantibody, a recombinant antibody, an antigen binding fragment or anycombination thereof. In some embodiments, the antibody is a humanizedmonoclonal antibody, or antigen binding fragment thereof, that binds toa GFRAL polypeptide (e.g., a cell surface-expressed or soluble GFRAL), aGFRAL fragment, or a GFRAL epitope.

The present disclosure also provides binding proteins such as anti-GFRALantibodies (i) that competitively block (e.g., in a dose-dependentmanner) an anti-GFRAL antibody provided herein from binding to a GFRALpolypeptide (e.g., a cell surface-expressed or soluble GFRAL), a GFRALfragment, or a GFRAL epitope and/or (ii) that bind to a GFRAL epitopethat is bound by an anti-GFRAL antibody provided herein. In otherembodiments, the binding proteins such as anti-GFRAL antibodycompetitively block (e.g., in a dose-dependent manner) monoclonalantibody 25M22, 3P10, 8D8 or 5F12 described herein or a humanizedvariant thereof from binding to a GFRAL polypeptide (e.g., a cellsurface-expressed or soluble GFRAL protein), a GFRAL fragment, or aGFRAL epitope. In other embodiments, the binding proteins such asanti-GFRAL antibody bind to a GFRAL epitope that is bound (e.g.,recognized) by monoclonal antibody 25M22, 3P10, 8D8 or 5F12 describedherein or a humanized variant thereof.

The present disclosure also provides binding proteins, includingantibodies or fragments thereof, that (i) bind to an epitope of a GFRALprotein recognized by an antibody comprising a heavy chain variableregion having the amino acid sequence of SEQ ID NO: 3, 7, 11, or 15 anda light chain variable region having the amino acid sequence of SEQ IDNO: 4, 8, 12, or 16, respectively; or (ii) compete for the binding to aGFRAL protein with an antibody comprising a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 3, 7, 11, or 15 and a lightchain variable region having the amino acid sequence of SEQ ID NO: 4, 8,12, or 16, respectively. In some embodiments, binding proteins,including antibodies or fragments thereof, are provided herein that bindto a region, including an epitope, of one or more amino acids of a GFRALprotein (e.g., a human GFRAL protein). In some embodiments, bindingproteins, including antibodies or fragments thereof, bind to a region ofa GFRAL protein (e.g., one or more amino acid residues of anextracellular domain) including, for example, those that bind to: (i)domain 1 of a GFRAL protein (e.g., amino acid residues Q20 to S130 ofSEQ ID NO: 1797); (ii) domain 2 of a GFRAL protein (e.g., amino acidresidues C131 to C210 of SEQ ID NO: 1797); (iii) domain 3 of a GFRALprotein (e.g., amino acid residues C220 to C316 of SEQ ID NO: 1797); or(iv) an extracellular domain of a GFRAL protein (e.g., amino acidresidues Q20 to E351 of SEQ ID NO: 1797).

In some embodiments, binding proteins, including antibodies or fragmentsthereof, are provided herein that bind to a specific epitope (e.g., oneor more amino acid residues) of a GFRAL protein, including, for example,those that bind to: (i) an epitope of a GFRAL protein comprising atleast one of (e.g., one or more) amino acid residues SER156, GLN147,LEU148, ALA149, SER150, TYR151, LEU152, LYS153, ALA154, CYS155, PHE174,TYR175, GLU136, ALA137, CYS138, VAL139, GLY140, ASP141, VAL142, VAL143,CYS144, ASN145, ALA146, LEU186, CYS189, CYS191, ALA192, GLN193, SER194,ASP195, ILE196, PRO197, CYS198, GLN199, GLN200, SER201, LYS202, GLU203,ALA204, LEU205, HIS206, SER207, SER130, CYS131, LEU132, GLU133, VAL134,or ALA135 of a GFRAL protein (SEQ ID NO: 1797); (ii) an epitope of aGFRAL protein comprising at least one of (e.g., one or more) amino acidresidues LEU132, GLU133, VAL134, ALA135, GLU136, ALA137, CYS138, VAL139,GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, GLN147, LEU148,ALA149, SER150, TYR151, PHE174, TYR175, ALA169, ALA170, ILE171, ARG172,PHE173, GLN176, ASN177, ILE178, PRO179, PHE180, ASN181, ILE182, ALA183,GLN184, MET185, LEU186, ALA187, PHE188, or CYS189 of SEQ ID NO: 1797;(iii) an epitope of a GFRAL protein comprising at least one of (e.g.,one or more) amino acid residues LEU164, LYS208, VAL212, ASN213, MET214,VAL215, PRO216, PRO217, PRO218, THR219, CYS220, LEU221, VAL223, TRP245,LEU267, CYS269, GLN28, VAL289, GLN290, CYS291, THR292, CYS293, ARG294,THR295, ILE296, THR297, GLN298, SER299, GLU300, GLU301, SER302, LEU303,CYS304, LYS305, ILE306, PHE307, GLN308, HIS309, MET310, LEU311, HIS312,ARG313, LYS314, SER315, CYS316, or PHE317 of SEQ ID NO: 1797; (iv) anepitope of a GFRAL protein comprising at least one of (e.g., one ormore) amino acid residues CYS233, ARG234, ARG235, HIS236, TYR237,ARG238, THR239, PHE240, GLN241, SER242, LYS243, CYS244, TRP245, GLN246,ARG247, VAL248, THR249, ARG250, LYS251, CYS252, HIS253, GLU254, ASP255,GLU256, ASN257, CYS258, ILE259, SER260, THR261, LEU262, SER263, LYS264,ASP266, LEU267, THR268, SER272, ASP274, CYS275, ALA278, CYS269, SER270,SER302, LEU303, ILE306, HIS309, LEU311, MET310, SER315, or CYS316 of SEQID NO: 1797; (v) an epitope of a GFRAL protein comprising at least oneof (e.g., one or more) amino acid residues GLY140, LEU148, ALA149,ALA146, VAL142, ASN145, VAL139, ALA135, GLU136, LEU152, LEU132, SER201,ALA204, LEU205, LYS153, ILE196, PRO197, or GLN200 of SEQ ID NO: 1797;(vi) an epitope of a GFRAL protein comprising at least one of(e.g., oneor more) amino acid residues GLU136, ALA137, VAL139, GLY140, ASP141,VAL142, VAL143, CYS144, ASN145, ALA146, GLN147, PHE173, ASN177, ILE178,PRO179, ASN181, ILE182, or MET185 of SEQ ID NO: 1797; (vii) an epitopeof a GFRAL protein comprising at least one of (e.g., one or more) aminoacid residues GLN298 or GLU301 of SEQ ID NO: 1797; or (viii) an epitopeof a GFRAL protein comprising at least one of (e.g., one or more) aminoacid residues ARG234, ARG238 , GLN241, SER242, LYS243, TRP245, GLN246,THR249, ARG250, LYS251, CYS252, HIS253, ASP255, ASN257, CYS258, SER260,THR261, or LEU262 of SEQ ID NO: 1797. Such antibodies provided abovecan, in some embodiments, inhibit GDF15-induced signaling and/orsignaling or activation of a GFRAL/GDF15 or RET/GFRAL/GDF15 receptorcomplex, for example, in a cell that expresses a GFRAL protein.Additionally, in some embodiments, the antibody is a monoclonalantibody, for example, a humanized, human or chimeric antibody.

In some embodiments, the binding proteins such as anti-GFRAL antibodiesprovided herein are conjugated or recombinantly linked to a diagnosticagent, a detectable agent (e.g., a radioisotope, an enzyme, afluorescent compound, a bioluminescent compound or a chemiluminescentcompound). In some embodiments, the binding proteins such as anti-GFRALantibodies provided herein are used (e.g., administered) with atherapeutic agent. In some aspects, the therapeutic agent is a drug,including one or more drugs such as an inhibitor of Activin-A, aninhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, aghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFαinhibitor, an IL-Ia inhibitor, a myostatin inhibitor, a beta-blocker, amelanocortin peptide inhibitor, a melanocortin receptor inhibitor, or ananti-cancer agent.

In certain embodiments, compositions are provided comprising a bindingprotein such as an anti-GFRAL antibody described herein. Also providedherein are pharmaceutical compositions comprising a binding protein suchas an GFRAL antibody as described herein.

The present disclosure also provides isolated nucleic acid moleculesencoding an immunoglobulin heavy chain, an immunoglobulin light chain,VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and/or VL CDR3 of binding proteins (e.g., anti-GFRAL antibodies) thatbind to a GFRAL polypeptide, a GFRAL polypeptide fragment, or a GFRALepitope (e.g., one or more amino acids of a GFRAL protein, including ofan extracellular domain of a GFRAL protein). In some embodiments, thenucleic acid molecule encodes a VH region, VL region, VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibodydesignated as 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5,2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6,P1H8, or P8G4 as described herein, or a humanized variant thereof. Insome embodiments, the nucleic acid molecule further encodes a scaffoldregion or a framework region, including VH FR1, VH FR2, VH FR3, VH FR4,VL FR1, VL FR2, VL FR3, and/or VL FR4 of a human immunoglobulin aminoacid sequence or a variant thereof. Also provided herein are vectors andhost cells comprising the nucleic acid molecules encoding a bindingprotein such as anti-GFRAL antibody, as well as methods of producing abinding protein such as an anti-GFRAL antibody by culturing the hostcells provided herein under conditions that promote the production of abinding protein such as an anti-GFRAL antibody.

The present disclosure also provides methods of treating, preventing oralleviating a GFRAL-mediated disease, disorder, or condition, includinga GDF15-mediated disease, disorder or condition, (e.g., one or moresymptoms) comprising administering to a subject a therapeuticallyeffective amount of a binding protein such as an anti-GFRAL antibodyprovided herein, including a subject in need thereof, thereby treating,preventing or alleviating the disease, disorder or condition. In someembodiments, the disease, disorder or condition is caused by orotherwise associated with a GDF15 protein (e.g., a human GDF15 protein)and/or a GFRAL protein (e.g., a human GFRAL protein), such as thoserelated to GDF15-induced signaling in a subject. In certain embodiments,the disease, disorder, or condition is treatable by reducing theoccurrence, frequency or severity of cachexia, sarcopenia, or musclewasting, bone wasting or involuntary loss of body weight. In certainembodiments, the disease, disorder, or condition is cachexia. In certainembodiments, the disease, disorder, or condition is a cancer. In certainembodiments, the disease, disorder, or condition is a cardiovasculardisease. In certain embodiments, the disease, disorder, or condition isa chronic inflammatory disease (e.g., chronic renal disease, chronicobstructive pulmonary disease). In certain embodiments, the disease,disorder, or condition is a cancer that has decreased sensitivity to(e.g., resistance to) a chemotherapeutic agent (e.g., an anti-tumorantibody such as trastuzumab) that is induced by or related to a GDF15protein, including elevated levels of GDF15.

In some embodiments, the disease, disorder or condition is or is relatedto cachexia, sarcopenia, muscle wasting or loss of muscle mass, bonewasting, involuntary loss of body weight (e.g., body weight lossassociated with or due to a disease, disorder, or condition). In someembodiments, the disease, disorder or condition is selected from thegroup of underlying diseases associated with cachexia including, but arenot limited to, cancer, chronic renal disease, chronic obstructivepulmonary disease, AIDS, tuberculosis, chronic inflammatory diseases,sepsis and other forms of systemic inflammation, muscle wasting, such asmuscular dystrophy, and the eating disorder known as anorexia nervosa.

In some embodiments, the methods of treating, preventing or amelioratinginclude methods of improving body weight gain or reducing body weightloss, or improving muscle mass gain or reducing muscle mass loss. Insome embodiments, the methods of treating, preventing or amelioratingresult in improved methods of treating cancer, by preventing, minimizingor reducing the occurrence, frequency or severity of cachexia,sarcopenia or muscle wasting, bone wasting or involuntary loss of bodyweight.

The present disclosure also provides methods for detecting GFRAL in asample comprising contacting the sample with a binding protein such asan anti-GFRAL antibody as described herein, that comprises a detectibleagent. In certain embodiments, the sample comprises a cell expressingGFRAL on its surface.

The present disclosure also provides kits comprising a binding proteinsuch as an anti-GFRAL antibody that binds to a GFRAL polypeptide, aGFRAL fragment or a GFRAL epitope as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment between various GFRAL proteins. SEQ IDNOS are noted in parenthesis and bold text.

FIG. 2 shows a sequence alignment between various GDF15 proteins. SEQ IDNOS are noted in parenthesis and bold text.

FIG. 3 depicts results of an experiment showing binding affinity ofexemplary anti-GFRAL antibodies 1C1 and 3P10 for a GFRAL protein.

FIG. 4A-4B show alignments of VH and VL sequences for exemplaryanti-GFRAL antibodies. SEQ ID NOS are noted in parenthesis and boldtext.

FIG. 5A-5F show alignments of VH and VL sequences for exemplaryanti-GFRAL antibodies that bind to domain 1 (FIGS. 5A-5B), domain 2(FIGS. 5C-5D), or domain 3 (FIGS. 5E-5F) of GFRAL. SEQ ID NOS are notedin parenthesis and bold text.

FIGS. 6A-6B show alignments of VH and VL sequences of humanized 1C1antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 7A-7B show alignments of VH and VL sequences of humanized 25M22antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 8A-8B show alignments of VH and VL sequences of humanized 17J16antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 9A-9B show alignments of VH and VL sequences of humanized 5F12antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 10A-10B show alignments of VH and VL sequences of humanized 3P10antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIG. 11 depicts results of a receptor antagonist assay using a humanizedGFRAL antibody.

FIG. 12 depicts results for an Elk1 reporter assay showing of humanizedantibodies.

FIGS. 13A-13C depict results of an experiment showing specificity of anexemplary humanized anti-GFRAL antibody.

FIGS. 14A-14B depict results of an experiment showing anti-GFRALantibody inhibition of GDF15-induced weight loss in DIO mice. For FIG.14A, from left to right in the figure, the administered treatment wasPBS, 3P10, 1C1, 17J16, 5A20, 25M22, 5F12, and 1MO3, respectively (ford1, d2 and d3, respectively). For FIG. 14B, from left to right in thefigure, the administered treatment was PBS, 8D8 and 12A3, respectively(for d1, d2 and d3, respectively).

FIG. 15 depicts results of an experiment showing anti-GFRAL antibodiesin a model of GDF15-induced weight loss.

FIG. 16 depicts results of an experiment showing the effects ofanti-GFRAL antibodies on food intake.

FIG. 17 depicts results of an experiment showing the effects ofanti-GFRAL antibodies on body weight in DIO mice.

FIG. 18 depicts results of an experiment showing the effects ofanti-GFRAL antibodies on food intake in DIO mice.

FIG. 19 depicts results of an experiment showing the effects ofanti-GFRAL antibodies on fat mass.

FIG. 20 depicts results of an experiment showing the effect ofanti-GFRAL antibodies on GDF15-induced loss of body weight and loss offat and lean mass in DIO m ice.

FIG. 21 depicts results of an experiment showing the effects ofanti-GFRAL antibodies on GDF15-induced increase in energy expenditureand GDF15-induced reduction in food intake in DIO mice.

FIG. 22 depicts results of an experiment showing the effects ofanti-GFRAL antibodies on GDF15-induced loss of body weight and loss offat and lean mass in lean m ice.

FIG. 23 depicts results of an experiment showing the effects anti-GFRALantibodies on GDF15-induced change in RER and GDF15-induced reduction infood intake in lean mice.

FIG. 24 depicts results of an experiment showing the effects of of ananti-GFRAL antibody (3P10) on body weight in lean mice.

FIG. 25 depicts results of an experiment showing the effects ofadministration of anti-GFRAL antibodies on food intake in lean mice.

FIGS. 26A-26C depict results of an experiment showing the effects ofdietary adenine.

FIG. 27A depicts results of an experiment showing the effects of dietaryadenine.

FIG. 27B depicts results of an experiment showing the effects of anexemplary an anti-GFRAL antibody (3P10) on mice with chronic kidneydamage.

FIG. 28 depicts results of an experiment showing the effects of anexemplary an anti-GFRAL antibody (3P10) on mice with chronic kidneydamage.

FIG. 29 depicts results of an experiment showing the effects of anexemplary humanized anti-GFRAL antibody (h3P10) of GDF15-induced bodyweight loss.

FIG. 30 shows an exemplary crystal of a complex ofa GFRAL protein and aGDF15 protein.

FIG. 31 illustrates an exemplary GFRAL electron density map.

FIG. 32 shows an exemplary ribbon diagram of a GFRAL/GDF15 complexformed in an asymmetric GFRAL/GDF15 crystal unit. GFRAL protein domainsD2 and D3 are indicated as GFRAL D2 and GFRAL D3.

FIG. 33 shows an exemplary ribbon diagram of a dimer of two GFRAL/GDF15complexes. The GFRAL protein domains D2 and D3 are indicated as GFRAL D2and GFRAL D3.

FIGS. 34A-34B show different surface representations of a dimer of twoGFRAL/GDF15 complexes.

FIG. 35 illustrates GFRAL amino acid residues interacting with GDF15amino residues.

FIGS. 36A-36D illustrate a GFRAL/GDF15 interface. The GFRAL proteindomains D2 and D3 are indicated as GFRAL D2 and GFRAL D3.

FIGS. 37A-37B show different aspects of a superposition of a GFRALprotein and GFRα1 depicted as ribbon diagrams.

FIGS. 38A-38D illustrate different aspects of the interaction of a GFRALprotein with a RET protein in a RET/GFRAL/GDF15 model.

FIGS. 39A-39B illustrate amino acid residues on the RET proteininterface of a GFRAL protein.

FIG. 40 illustrates exemplary crystals of a complex having a GFRALprotein, a 3P10 Fab and a 25M22 Fab produced under crystallizationconditions B11, D11, and H8.

FIG. 41 illustrates exemplary crystals of a complex having a GFRALprotein, a 8D8 Fab and a 5F12 Fab produced under crystallizationconditions C6, E11, and C2.

FIG. 42 illustrates exemplary electron density maps of 3P10 Fab and25M22 Fab CDR regions in the crystal structure of a GFRAL/3P10/25M22 Fabcomplex.

FIG. 43 illustrates an exemplary electron density of a GFRAL/8D8/5F12Fab complex.

FIG. 44 shows an exemplary ribbon diagram of a GFRAL/3P10/25M22 Fabcomplex formed in an asymmetric GFRAL/3P10/25M22 Fab complex crystalunit.

FIG. 45 shows alignments of 3P10 Fab and 25M22 Fab CDR sequences (toplines) with GFRAL amino acid residues (bottom lines) that are involvedin 3P10 Fab and 25M22 Fab binding. Residues involved in the GFRAL-Fabinteraction are boxed. For the 3P10 Fab, amino acid residues Q1 to S120of SEQ ID NO: 1824 are shown for the Hc and amino acid residues D1 toF120 of SEQ ID NO: 1825 are shown for the Lc. For the 25M22 Fab, aminoacid residues Q1 to S120 of SEQ ID NO: 1826 are shown for the Hc.

FIG. 46 shows a ribbon diagram illustrating the interaction of a GFRAL3P10 Fab epitope and a 3P10 Fab heavy chain CDR region.

FIG. 47 shows a ribbon diagram illustrating the interaction of a GFRAL3P10 Fab epitope and a 3P10 light chain CDR region.

FIG. 48 shows a ribbon diagram illustrating the interaction of a GFRAL25M22 epitope and a 25M22 Fab heavy chain CDR region.

FIG. 49 shows amino acid sequences for a GFRAL protein (residues S130 toN318 of SEQ ID NO: 1797) and 25M22 Fab heavy chain (HC) residues Q1 toP138 and G146 to P225 of SEQ ID NO:1826 and light chain (LC) residues D1to E218 of SEQ ID NO:1827. Residues having grey background and whitetext indicate core interaction interface amino acids for both the GFRALprotein and the 25M22 Fab. Residues on the Fab HC or LC having greybackground with black or white lettering indicate exemplary CDRsequences for the 25M22 Fab.

FIGS. 50A-50B illustrate core interaction interface amino acid residueson a GFRAL protein and on 25M22 Fab involved in the GFRAL/25M22 Fabinteraction.

FIG. 51 illustrates boundary interaction interface amino acid residueson a GFRAL protein involved in the GFRAL/25M22 Fab interaction.

FIG. 52 shows exemplary side views of a ribbon diagram illustratingoverlapping 25M22 Fab and GDF15 epitopes on a GFRAL protein asspace-filled surface models (core interaction interface amino acids).

FIG. 53 shows a top view of a ribbon diagram illustrating overlapping25M22 Fab and GDF15 epitopes on a GFRAL protein as space-filled surfacemodels (core interaction interface amino acids).

FIG. 54 shows amino acid sequences for a GFRAL protein (residues S130 toN318 of SEQ ID NO: 1797) and 3P10 Fab heavy chain (HC) residues Q1 toA130 and G138 to C221 of SEQ ID NO:1824 and light chain (LC) residues D1to C218 of SEQ ID NO:1825. Residues having grey background and whitetext indicate core interaction interface amino acids for both the GFRALprotein and the 3P10 Fab. Residues on the Fab HC or LC having greybackground with black or white lettering indicate exemplary CDRsequences for the 3P10 Fab.

FIGS. 55A-55B show a ribbon diagram illustrating a crystal structure ofa GFRAL/3P10 Fab complex. Interaction interface residues are shown asstick models (FIG. 55A) or space-filled surface models (FIG. 55B).

FIGS. 56A-56B illustrates interface residues of a GFRAL 3P10 Fab epitopeas space-filled surface models in a ribbon diagram of GFRAL. FIG. 56Ashows core interaction interface residues and FIG. 56B shows boundaryinteraction interface residues.

FIG. 57 illustrates overlapping residues of a GFRAL protein that bind to3P10 Fab and residues of a GFRALprotein that bind to a RET protein asspace-filled surface models on a ribbon diagram of a GFRALprotein.

FIG. 58 illustrates the combined coverage of boundary interactioninterface residues of a GFRAL protein that bind to 3P10 Fab and 25M22Fab.

FIG. 59 shows an exemplary ribbon diagram of a GFRAL/8D8/5F12 Fabcomplex formed in an asymmetric GFRAL/8D8/5F12 Fab complex crystal unit.

FIG. 60 shows an exemplary ribbon diagram of the 8D8 Fab and 5F12 Fabbinding sites on a GFRAL protein. Residues on the GFRAL protein that areimportant for Fab binding are shown as stick models.

FIG. 61 shows amino acid sequences for a GFRAL protein (residues S130 toN318 of SEQ ID NO: 1797) and 8D8 Fab heavy chain (HC) residues Q1 toK217 of SEQ ID NO:1828 and light chain (LC) residues D1 to R211 of SEQID NO:1829. Residues having grey background and white text indicate coreinteraction interface amino acids for both the GFRAL protein and the 8D8Fab. Residues on the Fab HC or LC having grey background with black orwhite lettering indicate exemplary CDR sequences for the 8D8 Fab.

FIGS. 62A, 62B, 62C and 62D illustrate core and boundary amino acidresidues in a GFRAL/8D8 Fab interaction interface.

FIG. 63A, 63B, 63C and 63D illustrate the core and boundary amino acidresidues in a GFRAL/5F12 Fab interaction interface.

FIG. 64 shows amino acid sequences for a GFRAL (residues S130 to N318 ofSEQ ID NO: 1797) and 5F12 Fab heavy chain (HC) residues Q1 to K223 ofSEQ ID NO:1830 and light chain (LC) residues N1 to E217 of SEQ IDNO:1831. Residues having grey background and white text indicate coreinteraction interface amino acids for both the GFRAL protein and the5F12 Fab. Residues on the Fab HC or LC having grey background with blackor white lettering indicate exemplary CDR sequences for the 5F12 Fab.

DETAILED DESCRIPTION

Binding proteins, such as antibodies that bind a GFRAL protein,including a human GFRAL protein, are provided herein. A unique propertyof such binding proteins, including antibodies disclosed herein, istheir antagonistic nature, including the ability to inhibit an effect ofa GDF15 protein and/or to inhibit binding of a GDF15 protein to a GFRALprotein or inhibit binding of a RET protein to a GFRAL protein,including wherein the inhibition of binding reduces (e.g., blocks) GDF15signaling. Remarkably and specifically, binding proteins such asantibodies to a GFRAL protein disclosed herein (i) bind to a GFRALprotein, (ii) inhibit binding of a GDF15 protein to a GFRAL protein,and/or (iii) inhibit binding of a RET protein to a GFRAL protein,including blocking the formation of a GDF15/GFRAL protein complex or aGDF15/GFRAL/RET protein complex or GDF15 signaling, including, forexample, as measured by several in vitro cell-based assays. Such assaysmay include (1) a ELK1-luciferase reporter assay (see, e.g., Example 3);and/or (2) ERK-phosphorylation assay in U2OS cells (see, e.g., Example4). Binding proteins such as anti-GFRAL antibodies, as described herein,therefore are expected to inhibit GDF15 activities in vivo (e.g.,related to the signaling function of GDF15). This property makes thedisclosed binding proteins, including anti-GFRAL antibodies, viabletherapeutics for the treatment of a disease, disorder or condition thatis caused by or otherwise associated with a GDF15 protein (e.g., a humanGDF15 protein) and/or a GFRAL protein (e.g., a human GFRAL protein),such as those related to GDF15-induced signaling in a subject.

The binding proteins, such as antibodies that bind a GFRAL protein, thatare provided herein share the common feature of antagonizing the bindingof (i) a GDF15 protein to a GFRAL protein and/or (ii) a RET protein to aGFRAL protein. The anti-GFRAL antibodies provided herein includehumanized anti-GFRAL antibodies, including humanized anti-GFRALantibodies derived from or based on 1C1, 3P10, 12A3, 5F12, 5A20, 8D8,17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2,6G9, 2B11, 1A3, P1B6, P1H8, and/or P8G4 having CDR sequences asdescribed in Tables 1-24 or FIGS. 4-10. Such anti-GFRAL antibodies,including humanized anti-GFRAL antibodies, bind to a specific domain ofa GFRAL protein including, for example, those that bind to: (i) domain 1of a GFRAL protein (e.g., amino acid residues Q20 to S130 of SEQ ID NO:1797); (ii) domain 2 of a GFRAL protein (e.g., amino acid residues C131to C210 of SEQ ID NO: 1797); (iii) domain 3 of a GFRAL protein (e.g.,amino acid residues C220 to C316 of SEQ ID NO: 1797); or (iv) anextracellular domain of a GFRAL protein (e.g., amino acid residues Q20to E351 of SEQ ID NO: 1797).

In some embodiments of the present disclosure, the binding proteins suchas anti-GFRAL antibodies may comprise immunoglobulin variable regionswhich comprise one or more complementary determining regions (CDRs) asdescribed in Tables 1-24. In such binding proteins (e.g., anti-GFRALantibodies), the CDRs may be joined with one or more scaffold regions orframework regions, which orient(s) the CDR(s) such that the properantigen binding properties of the CDR(s) is achieved. Such bindingproteins, including anti-GFRAL antibodies as described herein, caninhibit (e.g., block) the interaction (i) between a GDF15 protein and aGFRAL protein and/or (ii) between a RET protein and a GFRAL protein.Such binding proteins, including anti-GFRAL antibodies as describedherein, can inhibit (e.g., block) GDF15 signaling.

In some embodiments of the present disclosure, the binding proteins suchas anti-GFRAL antibodies bind to: (i) an epitope of a GFRAL proteincomprising at least one of (e.g., one or more) amino acid residuesSER156, GLN147, LEU148, ALA149, SER150, TYR151, LEU152, LYS153, ALA154,CYS155, PHE174, TYR175, GLU136, ALA137, CYS138, VAL139, GLY140, ASP141,VAL142, VAL143, CYS144, ASN145, ALA146, LEU186, CYS189, CYS191, ALA192,GLN193, SER194, ASP195, ILE196, PRO197, CYS198, GLN199, GLN200, SER201,LYS202, GLU203, ALA204, LEU205, HIS206, SER207, SER130, CYS131, LEU132,GLU133, VAL134, or ALA135 of a GFRAL protein (SEQ ID NO: 1797); (ii) anepitope of a GFRAL protein comprising at least one of (e.g., one ormore) amino acid residues LEU132, GLU133, VAL134, ALA135, GLU136,ALA137, CYS138, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145,ALA146, GLN147, LEU148, ALA149, SER150, TYR151, PHE174, TYR175, ALA169,ALA170, ILE171, ARG172, PHE173, GLN176, ASN177, ILE178, PRO179, PHE180,ASN181, ILE182, ALA183, GLN184, MET185, LEU186, ALA187, PHE188, orCYS189 of SEQ ID NO: 1797; (iii) an epitope of a GFRAL proteincomprising at least one of (e.g., one or more) amino acid residuesLEU164, LYS208, VAL212, ASN213, MET214, VAL215, PRO216, PRO217, PRO218,THR219, CYS220, LEU221, VAL223, TRP245, LEU267, CYS269, GLN28, VAL289,GLN290, CYS291, THR292, CYS293, ARG294, THR295, ILE296, THR297, GLN298,SER299, GLU300, GLU301, SER302, LEU303, CYS304, LYS305, ILE306, PHE307,GLN308, HIS309, MET310, LEU311, HIS312, ARG313, LYS314, SER315, CYS316,or PHE317 of SEQ ID NO: 1797; (iv) an epitope of a GFRAL proteincomprising at least one of (e.g., one or more) amino acid residuesCYS233, ARG234, ARG235, HIS236, TYR237, ARG238, THR239, PHE240, GLN241,SER242, LYS243, CYS244, TRP245, GLN246, ARG247, VAL248, THR249, ARG250,LYS251, CYS252, HIS253, GLU254, ASP255, GLU256, ASN257, CYS258, ILE259,SER260, THR261, LEU262, SER263, LYS264, ASP266, LEU267, THR268, SER272,ASP274, CYS275, ALA278, CYS269, SER270, SER302, LEU303, ILE306, HIS309,LEU311, MET310, SER315, or CYS316 of SEQ ID NO: 1797; (v) an epitope ofa GFRAL protein comprising at least one of (e.g., one or more) aminoacid residues GLY140, LEU148, ALA149, ALA146, VAL142, ASN145, VAL139,ALA135, GLU136, LEU152, LEU132, SER201, ALA204, LEU205, LYS153, ILE196,PRO197, or GLN200 of SEQ ID NO: 1797; (vi) an epitope of a GFRAL proteincomprising at least one of(e.g., one or more) amino acid residuesGLU136, ALA137, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145,ALA146, GLN147, PHE173, ASN177, ILE178, PRO179, ASN181, ILE182, orMET185 of SEQ ID NO: 1797; (vii) an epitope of a GFRAL proteincomprising at least one of (e.g., one or more) amino acid residuesGLN298 or GLU301 of SEQ ID NO: 1797; or (viii) an epitope of a GFRALprotein comprising at least one of (e.g., one or more) amino acidresidues ARG234, ARG238 , GLN241, SER242, LYS243, TRP245, GLN246,THR249, ARG250, LYS251, CYS252, HIS253, ASP255, ASN257, CYS258, SER260,THR261, or LEU262 of SEQ ID NO: 1797. Such antibodies provided abovecan, in some embodiments, inhibit GDF15-induced signaling and/orsignaling or activation of a GFRAL/GDF15 or RET/GFRAL/GDF15 receptorcomplex, for example, in a cell that expresses a GFRAL protein.Additionally, in some embodiments of the present disclosure, theantibody is a monoclonal antibody, for example, a humanized antibody.

General Techniques

Techniques and procedures described or referenced herein include thosethat are generally well understood and/or commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized methodologies described in Sambrook et al.,Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); TherapeuticMonoclonal Antibodies: From Bench to Clinic, Z. An, ed, Wiley, HobokenN.J. (2009); Monoclonal Antibodies: Methods and Protocols, M. Albitar,ed., Humana Press, Totawa, N.J. (2010); and Antibody Engineering, 2ndEd., Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag,Heidelberg, 2010.

Terminology

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art. For purposes of interpreting thisspecification, the following description of terms will apply andwhenever appropriate, terms used in the singular will also include theplural and vice versa. All patents, applications, published applicationsand other publications are incorporated by reference in their entirety.In the event that any description of terms set forth conflicts with anydocument incorporated herein by reference, the description of term setforth below shall control.

The term “GDNF Family Receptor Alpha Like” “growth differentiationfactor 15 receptor,” “GFRAL” or “GFRAL protein” and similar terms refersto a polypeptide (“polypeptide,” and “protein” are used interchangeablyherein) or any native GFRAL from any vertebrate source, includingmammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs,and rodents (e.g., mice and rats), unless otherwise indicated, and, incertain embodiments, includes related GFRAL polypeptides, including SNPvariants thereof. GFRAL is also known in the art as “C6orf144,”“Chromosome 6 Open reading Frame 144,” BA360D14.1″ “IVFI9356,” and“UNQ9356.”

The amino acid sequence of a full-length precursor human GFRAL isprovided below, which includes a signal peptide sequence (underlined andlowercase residues):

(SEQ ID NO: 1797) mivfiflamglsleneytsQTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL.

The amino acid sequence of a mature human GFRAL polypeptide is providedbelow:

(SEQ ID NO: 1798) QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL.

In some embodiments, GFRAL refers to a protein that is at least 55%identical to the amino acid sequence of mature human GFRAL (SEQ ID NO:1798). Binding proteins, such as anti-GFRAL antibodies as disclosedherein, can bind GFRAL and/or modulate signaling, as described herein.In certain embodiments, antibodies described herein bind to human GFRAL.

Human GFRAL has an extracellular domain (e.g., residues 20-351 of SEQ IDNO: 1797), a transmembrane domain (e.g., residues 352-371 of SEQ ID NO:1797) and a cytoplasmic domain (e.g., residues 372-394 of SEQ ID NO:1797).

A nucleic acid sequence encoding a precursor GFRAL polypeptide isprovided below:

(SEQ ID NO: 1799) TTATTCTGGACAGTTACTCTTAAGAAAGTTGTCAGAAGAAACGCATCTGCCTTTTTTTCCAGGTGAACTGCCGTGAGTTGTCCAGCATGATAGTGTTTATTTTCTTGGCTATGGGGTTAAGCTTGGAAAATGAATACACTTCCCAAACCAATAATTGCACATATTTAAGAGAGCAATGCTTACGTGATGCAAATGGATGTAAACATGCTTGGAGAGTAATGGAAGATGCCTGCAATGATTCAGATCCAGGTGACCCCTGCAAGATGAGGAATTCATCATACTGTAACCTGAGTATCCAGTACTTAGTGGAAAGCAATTTCCAATTTAAAGAGTGTCTTTGCACTGATGACTTCTATTGTACTGTGAACAAACTGCTTGGAAAAAAATGTATCAATAAATCAGATAACGTGAAAGAGGATAAATTCAAATGGAATCTAACTACACGTTCCCATCATGGATTCAAAGGGATGTGGTCCTGTTTGGAAGTGGCAGAGGCATGTGTAGGGGATGTGGTCTGTAATGCACAGTTGGCCTCTTACCTTAAAGCTTGCTCAGCAAATGGAAATCCGTGTGATCTGAAACAGTGCCAAGCAGCCATACGGTTCTTCTATCAAAATATACCTTTTAACATTGCCCAGATGTTGGCTTTTTGTGACTGTGCTCAATCTGATATACCTTGTCAGCAGTCCAAAGAAGCTCTTCACAGCAAGACATGTGCAGTGAACATGGTTCCACCCCCTACTTGCCTCAGTGTAATTCGCAGCTGCCAAAATGATGAATTATGCAGGAGGCACTATAGAACATTTCAGTCAAAATGCTGGCAGCGTGTGACTAGAAAGTGCCATGAAGATGAGAATTGCATTAGCACCTTAAGCAAACAGGACCTCACTTGTTCAGGAAGTGATGACTGCAAAGCTGCTTACATAGATATCCTTGGGACGGTCCTTCAAGTGCAATGTACCTGTAGGACCATTACACAAAGTGAGGAATCTTTGTGTAAGATTTTCCAGCACATGCTTCATAGAAAATCATGTTTCAATTATCCAACCCTGTCTAATGTCAAAGGCATGGCATTGTATACAAGAAAACATGCAAACAAAATCACTTTAACTGGATTTCATTCCCCCTTCAATGGAGAAGTAATCTATGCTGCCATGTGCATGACAGTCACCTGTGGAATCCTTCTGTTGGTTATGGTCAAGCTTAGAACTTCCAGAATATCAAGTAAAGCAAGAGATCCTTCATCGATCCAAATACCTGGAGAACTCTGATTCATTAGGAGTCATGGACCTATAACAATCACTCTTTTCTCTGCTTTTCTTCTTTCCTCTTTTCTTCTCTCCTCTCCTCTCCTCTCTTCTCCTCTCCTCCCCTCCCCTCTCTGTTTCTTTTTCTTTTTCTTTTCTTTTTTGTGGCGGAGTTTTGCTCTTGTTGCCCAGGCTGCAGTACAATGGCTCAATCTCGGTTCACTGCAACCTCTGCCTCCAAGGTTCAAGTGATTTTCCTGCCTCAGCCTTCCCGAGTAGCTGGGATTACAGGTACCCGCCACCACGCCCAGCTAATTTTTTTGTATTTTTAGTAGAGATGGGGTTTTGCCAAATTGGCCAGGGTGGTCTCAAACTCCTGACCTCAGGTGATCCACCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCAACCACGTCAAGACAACAATCACTTTCTTTAAAGCAAATCCTACAGCTGGTCAACACCCTATTCCATCTGTCATCGAGAAAGAAAATGTTAAAATAGACTTAAAAATATTGCTTTGTTACATATAATAATATGGCATGATGATGTTATTTTTTTCTTAATACTCAAGAAAAAATATATGGTGGTATCTTTTACAACACTGGAACAGAAATAAAGTTT CCCTTGAAGGC.

“GFRAL” as used herein encompasses human GFRAL and variants thereof,including but not limited to orthologs thereof, such as murine GFRAL,rat GFRAL, cyno GFRAL, and the like. GFRAL is not TGFβ RII (NCBI Ref.Seqs.: NM_001024847.2 (GI:133908632); NM_003242.5 (GI:133908633)) ororthologs thereof. GFRAL is distinct from TGFβ RI (NCBI Ref. Seqs.:NP_001124388.1 (GI:195963412); NP_004603.1 (GI:4759226)) or orthologsthereof. In certain embodiments, GFRAL may be a protein having the aminoacid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1797. Suchexemplary GFRAL proteins include chimpanzee (99%), cynomolgus monkey(92%), giant panda (82%), dog (81%), cat (80%), pig (77%), bovine (75%),mouse (70%), rat (70%), Chinese hamster (65%), and platypus (59%), asshown in FIG. 1.

An amino acid sequence of a GFRAL protein from cynomolgus monkey (cyno),scientific name Macaca fascicularis, is provided below, which includes asignal peptide sequence (underlined residues):

(SEQ ID NO: 1800) mivliflalglsleneytsQTNNCTYLREQCLHDANGCKHAWRIMEDACNDSDPGDPCKMNNSSYCNLSIQYLVESNFRFKECLCTDDFYCTVNKLLGKECVNKSDNMREDKFKWNLTTHSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDVKHCQAAIRFFYQNIPFNIAQMLAFCDCSQSDIPCQQSKEALHSKPCALNMVPPPTCLNVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISALSKQDLTCSGSDDCKAAYIDILGTVLQVQCNCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKSMALYTRKHTNKITLTGFQSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSLSQVPGEL.

An encoding nucleic acid sequence of a cyno GFRAL protein is providedbelow:

(SEQ ID NO: 1801) ACCCACCAGAAAGAAGGAGCTCCAGACACATCTGAACGTCTGAAGGAAGAAACTCCCGACACACCATCTTTAAGAAATGTAACTCTCACTGCGAGGGTATGTGGCTTCATTCTTGAAGTCAGGGAGACCAAGAACCCACCAATTGCAGGCACACAAGGGGTCCTTATTTTATTCAGGTGAACAGCTGTGAGTTGTCCAGCATGATAGTGCTTATTTTCTTGGCTTTGGGGCTAAGCTTGGAAAATGAATACACTTCCCAAACCAATAATTGCACATATTTAAGAGAGCAATGCCTACATGATGCAAATGGATGTAAACATGCTTGGAGAATAATGGAAGATGCCTGCAATGATTCAGATCCAGGTGACCCCTGCAAGATGAATAATTCATCATACTGTAACCTGAGTATCCAGTACTTAGTGGAAAGCAATTTCCGATTTAAAGAGTGTCTTTGCACTGATGACTTCTATTGTACTGTGAACAAACTGCTTGGAAAAGAATGTGTCAATAAATCAGATAACATGAGAGAGGATAAATTCAAATGGAATCTAACTACACATTCCCATCATGGATTCAAAGGGATGTGGTCCTGTTTGGAAGTGGCAGAGGCATGTGTAGGGGATGTGGTCTGTAATGCACAGTTGGCCTCTTACCTTAAAGCTTGCTCAGCAAATGGAAATCCGTGTGATGTGAAACACTGCCAAGCAGCCATACGGTTCTTCTATCAAAATATACCTTTTAACATTGCCCAGATGTTGGCTTTTTGTGACTGTTCTCAATCTGATATACCTTGTCAGCAGTCCAAAGAAGCTCTTCACAGCAAGCCATGTGCACTGAACATGGTTCCACCCCCTACTTGCCTCAATGTAATTCGCAGCTGCCAAAATGATGAATTATGCAGGAGGCACTATAGAACATTTCAGTCAAAATGCTGGCAGCGTGTGACTAGAAAGTGCCATGAAGATGAGAATTGCATTAGCGCCTTAAGCAAACAGGACCTCACATGTTCAGGAAGTGATGACTGCAAAGCTGCTTACATAGATATCCTTGGGACAGTCCTTCAAGTGCAATGTAACTGTAGGACCATTACACAAAGTGAGGAATCTTTGTGCAAGATTTTCCAGCACATGCTTCATAGAAAATCATGTTTCAATTATCCAACCCTGTCTAATGTCAAAAGCATGGCATTGTATACAAGAAAACATACAAACAAAATCACTTTAACTGGATTTCAGTCCCCCTTCAATGGAGAAGTAATCTATGCTGCCATGTGCATGACAGTCACCTGTGGAATCCTTCTCTTGGTTATGGTCAAGCTTAGAACTTCCAGAATATCAAGTAAAGCAAGAGATCCTTCACTGAGCCAAGTACCTGGAGAACTCTGATTCATTAGGAGTCATGGACCCATAACAATCACTCCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCC.

An amino acid sequence of a GFRAL protein from mouse, scientific nameMus musculus, is provided below, which includes a signal peptidesequence (underlined residues):

(SEQ ID NO: 1802) mlvfiflavtlssenesssQTNDCAHLIQKCLIDANGCEQSWRSMEDTCLTPGDSCKINNSLHCNLSIQALVEKNFQFKECLCMDDLHCTVNKLFGKKCTNKTDNMEKDNKDKWNLTTTPFYHGFKQMQSCLEVTEACVGDVVCNAQLALYLKACSANGNLCDVKHCQAAIRFFYQNMPFNTAQMLAFCDCAQSDIPCQQSKETLHSKPCALNIVPPPTCLSVIHTCRNDELCRTHYRTFQTECWPHITGKCHEDETCISMLGKQDLTCSGSESCRAAFLGTFGTVLQVPCACRGVTQAEEHVCMIFQHMLHSKSCFNYPTPNVKDISSYEKKNSKEITLTGFNSFFNGELLYVVVCMAVTCGILFLVMLKLRIQSEKRDPSSIEIAGGVIIQ.

An encoding nucleic acid sequence of a mouse GFRAL protein is providedbelow:

(SEQ ID NO: 1803) AACAATTGAATTTGAATACAATTAGGAAAGTTCACAGCTCAAAACAAACTGGTGAGGAACAGCTGACACCAGAAGCTGACTCTAATTGGCTGGCTCTTAGGAAGCAAAACCTTTACACAGAAACTTCAGTTGGGATGTTGGTTGGTGTCAGTTCATCCGCCTTTCTCCCAGGGAGACCATCTTGAGTTGTCCAACATGCTAGTGTTCATTTTCCTGGCTGTTACGTTAAGCTCAGAAAATGAATCCTCTTCCCAAACAAATGATTGTGCACATTTAATACAGAAATGCTTGATTGATGCAAATGGCTGTGAGCAGTCATGGAGATCAATGGAAGACACCTGCCTTACTCCAGGTGACTCCTGCAAGATAAATAATTCACTACATTGTAACCTGAGTATCCAGGCTTTGGTGGAAAAAAATTTCCAATTTAAAGAGTGTCTTTGTATGGATGACCTCCACTGTACAGTAAACAAACTTTTTGGAAAAAAGTGCACCAATAAGACAGATAACATGGAAAAGGACAATAAAGATAAATGGAATCTAACTACTACTCCTTTCTATCATGGATTCAAACAGATGCAGTCTTGTTTGGAGGTGACAGAGGCGTGTGTAGGGGATGTGGTTTGTAATGCACAGTTGGCCCTTTACCTTAAAGCATGCTCAGCAAATGGAAATCTGTGTGATGTGAAACACTGCCAAGCAGCCATACGGTTCTTCTATCAAAATATGCCTTTTAACACTGCCCAGATGTTGGCTTTTTGTGACTGTGCTCAATCTGATATACCCTGTCAGCAATCCAAAGAAACTCTTCACAGCAAGCCATGTGCACTGAATATAGTTCCACCCCCCACTTGCCTCAGTGTAATTCACACTTGCCGAAATGATGAATTATGCAGGACACACTACCGAACATTCCAGACAGAATGCTGGCCCCACATAACTGGGAAGTGCCATGAAGATGAGACCTGCATTAGCATGTTAGGCAAGCAAGACCTTACTTGTTCTGGGAGTGAGAGCTGCAGGGCTGCCTTCCTAGGAACCTTTGGGACAGTCCTGCAAGTACCCTGTGCTTGCAGGGGCGTTACACAGGCTGAAGAACACGTGTGCATGATTTTCCAGCACATGCTTCATAGCAAATCGTGTTTCAATTACCCAACTCCTAATGTCAAAGACATTTCCTCATATGAAAAAAAGAATTCAAAAGAAATTACTCTGACTGGATTCAATTCTTTCTTCAATGGAGAACTACTCTATGTTGTTGTGTGCATGGCAGTTACCTGTGGAATTCTTTTCTTGGTGATGCTCAAGTTAAGGATACAAAGTGAAAAAAGAGATCCCTCATCCATCGAAATAGCTGGAGGTGTCATCATTCAGTGAGCTGCAGATCACTTACCAACCACATGTCTGTGTGACTAACCAATGGAAAATTACATTTGCCAATAACGCAATTTAAGATGGATTTGACAATATTTAGTCATTATATGTAACAGTGACTGGTACAGTAATATACCACAATGATCACAGATCTGTTTTTGTTTTTGTTTTTAATGTTTGAGTAAATACTTGTTGTGGTGTCATAACTAGTTGATAACATTTTCTTTAAAGACAACAGGTGTCATGTAAAATGTGACAAATTTGCTGGAAGACTATCAATCCACATATCAACTTCTATCTTATGGAACTAATCATAATTAGTGTGTGCAGTTTTCTGAACAAGGTTATAGTTTTCCATTAAGTTGGTAAAATTAAAATGCTAAGTAGAATATTGAGTATACTTGTTATTTATATATTCTTACTTAGTGTCCAATCATTAAACAAATTGGTAACATTGAACATATTTAGTTAGATGACTGCTTATGAAAATAAGAACTGACATCTTACAAATTTTATAATTTAAATAGTATTGAATTTTACTTTTTATTTGGTATGTTAAGATTCATAATATATAAAGCAGCTACATTGGTTGAGAAAAGTCAATGGTTACTCCAGTAATGATATACTTTGTGAATTTATTTATTTTTGCTAATTAATGATCCTGAATGTAATCATGATGAAATAAAAAAGACATACTTAAATTGCT.

An amino acid sequence of a GFRAL protein from rat, scientific nameRattus norvegicus, is provided below, which includes a signal peptidesequence (underlined residues):

(SEQ ID NO: 1804) mlvfiflavrlssenesssQTNDCAYFMRQCLTDTDGCKQSWRSMEDACLVSGDSCKINNPLPCNLSIQSLVEKHFQFKGCLCTDDLHCTVNKIFGKKCTNKTDSMKKDNKYKRNLTTPLYHDTGFKQMQSCLEVTEACVGDVVCNAQLALYLKACTANGNLCDVKHCQAAIRFFYQNMPFNTAQMLAFCDCAQSDIPCQQSKETLHSKPCALNVVPPPTCLSVIHTCRNDELCRTYYRTFQTECWPHVAGKCREDETCISMLGKQDLTCSGSDSCRAAYLGTFGTVLQVPCACRSITQGEEPLCMAFQHMLHSKSCFNYPTPNVKDISSYERKHSKEITLTGFNSPFSGELIYVVVCMVVTSGILSLVMLKLRIPSKKRDPAPIEIAGAVIIQ.

An encoding nucleic acid sequence of a rat GFRAL protein is providedbelow:

(SEQ ID NO: 1805) ACAAATGATTGTGCATATTTCATGCGGCAATGCTTGACTGATACAGATGGCTGTAAGCAGTCATGGAGATCAATGGAAGACGCCTGCCTTGTCTCAGGTGACTCCTGCAAGATAAATAATCCATTGCCTTGTAACCTGAGTATCCAGTCTTTGGTGGAAAAACATTTTCAATTTAAAGGGTGTCTTTGCACTGATGATCTCCACTGTACAGTAAACAAAATTTTTGGAAAAAAGTGCACCAATAAGACAGATAGCATGAAAAAAGATAATAAATACAAACGGAATCTAACTACTCCTTTATATCATGATACAGGATTCAAACAGATGCAGTCTTGTTTGGAAGTGACAGAGGCGTGTGTAGGGGATGTGGTTTGTAATGCACAGTTGGCCCTTTACCTTAAAGCATGCACAGCAAATGGAAATCTGTGTGATGTGAAACACTGCCAAGCGGCCATACGGTTCTTCTATCAAAATATGCCTTTTAACACTGCCCAGATGTTGGCTTTTTGTGACTGTGCTCAATCTGATATACCCTGTCAACAATCCAAAGAAACTCTTCACAGCAAGCCATGTGCACTGAACGTAGTTCCACCCCCCACTTGCCTCAGTGTAATTCACACTTGCCGAAATGATGAATTATGCAGGACATACTACCGAACATTCCAGACAGAATGCTGGCCCCATGTGGCTGGGAAGTGTCGTGAAGATGAGACCTGCATTAGTATGCTGGGCAAGCAAGACCTTACTTGTTCTGGGAGTGACAGCTGCAGGGCAGCCTACCTAGGAACCTTCGGGACAGTCCTTCAGGTGCCGTGTGCTTGCAGAAGCATCACACAGGGTGAAGAACCCTTGTGCATGGCTTTCCAGCACATGCTTCACAGCAAATCATGTTTCAATTACCCAACTCCTAATGTCAAAGACATTTCCTCATATGAAAGAAAGCATTCAAAAGAAATTACCCTGACTGGATTCAATTCTCCCTTCAGTGGAGAACTAATCTATGTTGTTGTGTGCATGGTAGTTACCAGCGGGATTCTTTCCTTGGTGATGCTCAAGCTAAGGATACCTAGTAAGAAAAGAGACCCCGCGCCCATCGAAATAGCTGGAGCTGTCATCATTCAGTGA.

A GFRAL protein or GFRAL also refers to a protein that has one or morealteration in the amino acid residues (e.g., at locations that are notconserved across variants and/or species) while retaining the conserveddomains and having a biological activity similar to thenaturally-occurring GFRAL. GFRAL may be encoded by nucleic acidsequences that vary in one or more bases from a naturally-occurring DNAsequence but still translate into an amino acid sequence thatcorresponds to the a naturally-occurring protein due to degeneracy ofthe genetic code. A GFRAL protein also refers to a protein that differsfrom the naturally-occurring sequences of GFRAL by one or moreconservative substitutions and/or tags and/or conjugates.

The term “GFRAL” or “GFRAL protein” encompasses “full-length”unprocessed GFRAL as well as any form of GFRAL that results fromprocessing in the cell. The term GFRAL or “GFRAL protein” also includes:allelic variants (e.g., SNP variants); splice variants; isoforms;fragments; derivatives; substitution, deletion, and insertion variants;fusion polypeptides; and interspecies homologs, preferably, which retainGFRAL activity and/or are sufficient to generate an anti-GFRAL immuneresponse. As those skilled in the art will appreciate, an anti-GFRALantibody provided herein can bind to a GFRAL protein, including a GFRALpolypeptide fragment, a GFRAL antigen, and/or a GFRAL epitope. Anepitope may be part of a larger GFRAL antigen, which may be part of alarger GFRAL polypeptide fragment, which, in turn, may be part of alarger GFRAL protein. A GFRAL protein may exist in a native or denaturedform. GFRAL proteins described herein may be isolated from a variety ofsources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods. A GFRAL protein maycomprise a polypeptide having the same amino acid sequence as acorresponding GFRAL polypeptide derived from nature. GFRAL proteinsencompass truncated or secreted forms of a GFRAL polypeptide (e.g., anextracellular domain sequence), variant forms (e.g., alternativelyspliced forms) and allelic variants of the polypeptide. GFRALpolypeptides described herein (e.g., human GFRAL) may be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods.

A GFRAL protein can lack at least 5, at least 10, up to at least 50 ormore amino acids relative to a naturally-occurring full-length GFRALpolypeptide. For example, the GFRAL protein may not contain the signalsequence based on the amino acid sequence of a naturally-occurring GFRALpolypeptide. A GFRAL protein may also contain the same or similarpost-translational modifications as a naturally-occurring GFRALpolypeptide or may not contain a post-translational modification. Forexample, the protein may have the same or similar glycosylation patternas those of a naturally-occurring GFRAL polypeptide or may contain noglycosylation. In other embodiments, the GFRL protein includes mutationsrelative to the sequence of naturally-occurring GFRAL protein thatintroduce a glycosylation site at a location not present in thenaturally-occurring GFRAL protein.

In certain embodiments, a GFRAL protein may be expressed by arecombinant cell genetically modified to express the GFRAL protein onits cell surface. The cell may be present in a composition that includesan isolated GDF15 protein. In certain cases, the cell may additionallyexpress a RET protein, for example the cell may express a RET proteinendogenously without being genetically modified to include an exogenoussequence encoding the RET protein. In other embodiments, the cell maynot express detectable levels of a RET protein and may be geneticallymodified to express a RET protein from an exogenous sequence.

Also disclosed herein are fragments of a GFRAL protein, such as GFRALfragments that lack an intracellular domain present in native GFRALprotein, or the intracellular domain and the transmembrane domainpresent in native GFRAL protein, such as a native GFRAL depicted inFIG. 1. As noted above, a fragment of a GFRAL protein may also lack asignal sequence present in the native GFRAL and may or may not include aheterologous signal sequence. The fragment may lack the intracellulardomain present in a native GFRAL protein but include the transmembranedomain.

The term “GFRAL-extracellular domain” (“GFRAL-ECD”) includes full-lengthGFRAL ECDs, GFRAL ECD fragments, and GFRAL ECD variants. As used herein,the term “GFRAL ECD” refers to a GFRAL polypeptide with or without asignal peptide that lacks the intracellular and/or transmembranedomains. In some embodiments, a GFRAL ECD refers to a protein having theamino acid sequence that is at least 70% identical to the amino acidsequence of a human full-length GFRAL ECD having the amino acidsequence:

(SEQ ID NO: 1806) QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGE.

The term “full-length GFRAL ECD”, as used herein, refers to a GFRAL ECDthat extends to the last amino acid of the extracellular domain, and mayor may not include an N-terminal signal peptide. However, it is notedthat “full-length GFRAL ECD” also encompasses a GFRAL-ECD that isextended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on theC-terminus to include amino acids residues of the transmembrane domainprovided that the polypeptide is soluble. In other words, such a GFRALECD lacks a sufficient length of a transmembrane domain such that it isnot anchored into a cell membrane. The phrase “full-length GFRAL ECD”also encompasses a GFRAL-ECD that is extended by 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acids on the N-terminus to include amino acids residuesof the signal peptide. In certain embodiments, a GFRAL ECD refers to acontiguous amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more identical to a contiguous amino acidsequence depicted in FIG. 1 and lacks at least 30, 33, 35, 40, 45, 50,or 55 amino acids or more at the C-terminus of the GFRAL sequencesdepicted in FIG. 1.

A GFRAL ECD is not an ECD of TGFβ RII (Acc. Nos.: NM_001024847.2;NM_003242.5) or orthologs thereof. GFRAL ECD is distinct from ECD ofTGFβ RI (Acc. Nos.: NP_001124388.1; NP _004603.1) or orthologs thereof.In certain embodiments, a GFRAL ECD may be a protein having the aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more identical to SEQ ID NO: 1806.

As used herein, the term “GFRAL ECD fragment” refers to a GFRAL ECDhaving one or more residues deleted from the N and/or C terminus of thefull-length ECD and that retains the ability to bind to GDF15. In someinstances, a GFRAL ECD fragment may or may not include an N-terminalsignal peptide. In some instances, a GFRAL ECD fragment is a human GFRALECD fragment that lacks 1, 5, 10, 15, 16, 17, 18, or 19 residues presentat the N-terminus of the sequence:

(SEQ ID NO: 1807) MIVFIFLAMGLSLENEYTSQTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITL TGFHSPFNGE

Another exemplary GFRAL ECD fragment comprises the following amino acidsequence, which corresponds to Q20 to C316 of a full-length humanprecursor GFRAL protein:

(SEQ ID NO: 1808) QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQH MLHRKSC

Yet another exemplary GFRAL ECD fragment comprises the following aminoacid sequence, which corresponds to W115 to E351 of a full-length humanprecursor GFRAL protein:

(SEQ ID NO: 1809) WNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGE.The above exemplary GFRAL ECD fragment was used in various methods asdescribed in the Examples, including to produce a crystal of a complexcomprising a GFRAL protein and a GDF15 protein or a GFRAL protein and anexemplary anti-GFRAL antibody.

Within a GFRAL protein or GFRAL ECD there are three domains—domain 1(D1), domain 2 (D2) and domain 3 (D3). In some embodiments, the aminoacid sequence of an exempalry D1 domain are residues Q20 to S130 of SEQID NO: 1797. In some embodiments, the amino acid sequence of anexemplary D2 domain are residues C131 to C210 of SEQ ID NO: 1797. Insome embodiments, the amino acid sequence of an exemplary D3 domain areresidues C220 to C316 of SEQ ID NO: 1797. Certain properties of a GFRALprotein can be attributed to the activity and/or binding of thesedomains, including within the ECD. For example, as described herein,amino acid residues within D2 are identified as being core interactioninterface amino acids and/or boundary interaction interface amino acidsfor a GFRAL protein binding to a GDF15 protein. Likewise, as describedherein, amino acid residues within D3 are identified as being coreinteraction interface amino acids and/or boundary interaction interfaceamino acids for a GFRAL protein binding to a RET protein.

The term “core interaction interface amino acid” or grammaticalequivalent thereof refers to an amino acid residue of a given proteinthat has at least one atom within less or equal to 4.5 Å from aninteracting protein (e.g., an amino acid of a GFRAL protein thatinteracts with a GDF15 protein or a RET protein). A distance of 4.5 Åallows for atoms within a van der Waals radius plus a possiblewater-mediated hydrogen bond to form a bond with the interactingprotein.

The term “boundary interaction interface amino acid” or grammaticalequivalent thereof refers to an amino acid residue of a given proteinthat has at least one atom within less than or equal to 5 Å from a coreinterface amino acid on the given protein (e.g., an amino acid of aGFRAL protein that is within 5 Å of a core interaction interface aminoacid of a GFRAL proteion that interacts with a GDF15 protein or a RETprotein). A distance of less than or equal to 5 Å allows proteinsbinding to residues less than 5 Å away from core interaction interfaceamino acids on a given protein to be within the van der Waals radius ofan interacting protein.

As used herein, the term “GFRAL ECD variants” refers to GFRAL ECDs thatcontain amino acid additions, deletions, or substitutions and thatremain capable of binding to GDF15. Such variants may be at least 80%,85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to a parent GFRAL ECD.

“Growth differentiation factor 15” or “GDF15,” also known in the art asMIC-1 (macrophage inhibitory cytokine-1), PDF (prostate differentiationfactor), PLAB (placental bone morphogenetic protein), NAG-1(non-steroidal anti-inflammatory drugs (NSAIDs) activated gene), TGF-PL,and PTGFB, is a member of the transforming growth factor β (TGF-β)super-family. GDF15, which is synthesized as a 62 kDa intracellularprecursor protein that is subsequently cleaved by a furin-like protease,is secreted as a 25 kDa disulfide-linked protein (see, e.g., Fairlie etal., J. Leukoc. Biol 65:2-5 (1999)). GDF15 mRNA is seen in severaltissues, including liver, kidney, pancreas, colon and placenta, andGDF15 expression in liver can be significantly up-regulated duringinjury of organs such as the liver, kidneys, heart and lungs.

The GDF15 precursor is a 308 amino acid polypeptide (NCBI Ref. Seq.NP_004855.2; GI:153792495) containing a 29 amino acid signal peptide, a167 amino acid pro-domain, and a mature domain of 112 amino acids whichis excised from the pro-domain by furin-like proteases.

An amino acid sequence of a precursor human GDF15 polypeptide isprovided below:

(SEQ ID NO: 1810) MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG VSLQTYDDLLAKDCHCISuch a 308-amino acid GDF15 polypeptide is referred to as a“full-length” GDF15 polypeptide; a 112-amino acid GDF15 polypeptide(amino acids 197-308 of “full-length” GDF15) is a “mature” GDF15polypeptide.

“GDF15” as used herein includes a protein having an amino acid sequencethat is at least 65% identical to the amino acid sequence of a maturehuman GDF15 polypeptide. An amino acid sequence of a mature human GDF15polypeptide is provided below:

(SEQ ID NO: 1811) ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV SLQTYDDLLAKDCHCI

The above exemplary mature human GDF15 was used in the various methodsdescribed as in the Examples, including to produce a crystal of acomplex comprising a GFRAL protein and a GDF15 protein.

Unless otherwise indicated, the term “GDF15” refers to a 112 amino acidmature human sequence (e.g., SEQ ID NO: 1811). In addition, numericalreferences to particular GDF15 residues refer to a 112 amino acid maturesequence (e.g., residue 1 is Ala (A), and residue 112 is IIe (I) of SEQID NO: 1811). For example, while a GDF15 precursor amino acid sequencepredicts three excision sites, resulting in three putative forms of“mature” human GDF15 (e.g., 110, 112 and 115 amino acids), the 112 aminoacid mature sequence is accepted as being correct.

Within the context of the present disclosure, “GDF15” or “GDF15 protein”includes GDF15 orthologs, and modified forms thereof, from othermammalian species, and their use, including mouse (NP_035949;GI:170784848), chimpanzee (XP_009433302.1; GI:694973734), orangutan(XP_009251261.1 GI:686757768), Rhesus monkey (EHH29815; GI:355703324),giant panda (XP_002912774; GI:301753921), gibbon (XP_004089328.1;GI:441627981), guinea pig (XP_003465238; GI:348558868), ferret(AER98997; GI:355689945), cow (NP_001193227; GI:329664989), pig(NP_001167527; GI:291291599), dog (XP_541938; GI:57101740) and platypus(Ornithorhynchus anatinus; AFV61279; GI:410111209). Such exemplary GDF15proteins are shown in FIG. 2, which includes an alignment of the variousexemplary GDF15 proteins. A mature form of human GDF15 has approximately67% amino acid identity to the mouse ortholog.

“RET,” also known in the art as Ret Proto-Oncogene, Cadherin-RelatedFamily Member 16, Rearranged During Transfection, RET Receptor TyrosineKinase, Cadherin Family Member 12, Proto-Oncogene C-Ret, EC 2.7.10.1,CDHF12, CDHR16, RET51, PTC, Hydroxyaryl-Protein Kinase, RET TransformingSequence, and Receptor Tyrosine Kinase, is one of the receptor tyrosinekinases, cell-surface molecules that transduce signals for cell growthand differentiation. RET acts as a co-receptor and is known as a primarysignaling receptor for glial-cell-line-derived neurotrophic factor(GDNF) ligands (in human, GDNF, artemin, neurturin, and persephin) whenbound to members of the GDNF receptor alpha (GFRα) co-receptors. A RETprotein (e.g., a RET-ECD) comprises 4 consecutive cadherin-like domains(CLD1-CLD4) followed by a membrane proximal cystine rich domain (CRD).As disclosed herein, a RET protein is a co-receptor with a GFRAL proteinand a GDF15 protein (e.g., acting as a co-receptor with a RET protein).A receptor complex, as described herein, includes a GFRAL protein, suchas a RET/GFRAL complex, a GFRAL/GDF15 complex, and a RET/GFRAL/GDF15complex.

As used herein, “Ret” or “RET” refers to a protein having the amino acidsequence that is at least 75% identical, e.g., 77%, 79%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence ofSEQ ID NO: 1813. RET is distinct from TGFβ RI and TGFβ RII. SEQ ID NO:1812 is the sequence of a mature human RET9 that lacks a signal peptide:

(SEQ ID NO: 1812) KVALGLYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPEDIQDPLCDELCRTVIAAAVLFSFIVSVLLSAFCIHCYHKFAHKPPISSAEMTFRRPAQAFPVSYSSSGARRPSLDSMENQVSVDAFKILEDPKWEFPRKNLVLGKTLGEGEFGKVVKATAFHLKGRAGYTTVAVKMLKENASPSELRDLLSEFNVLKQVNHPHVIKLYGACSQDGPLLLIVEYAKYGSLRGFLRESRKVGPGYLGSGGSRNSSSLDHPDERALTMGDLISFAWQISQGMQYLAEMKLVHRDLAARNILVAEGRKMKISDFGLSRDVYEEDSYVKRSQGRIPVKWMAIESLFDHIYTTQSDVWSFGVLLWEIVTLGGNPYPGIPPERLFNLLKTGHRMERPDNCSEEMYRLMLQCWKQEPDKRPVFADISKDLEKMMVKRRDYLDLAASTPSDSLIYDDGLSEEETPLVDCNNAPLPRALPSTWIENKLYGRISHAFTRF

The amino acid sequence of a full-length precursor human RET protein isprovided below, which includes a signal peptide sequence (underlined andlowercase residues):

(SEQ ID NO: 1813) makatsgaaglrlllllllpllgkvalgLYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPEDIQDPLCDELCRTVIAAAVLFSFIVSVLLSAFCIHCYHKFAHKPPISSAEMTFRRPAQAFPVSYSSSGARRPSLDSMENQVSVDAFKILEDPKWEFPRKNLVLGKTLGEGEFGKVVKATAFHLKGRAGYTTVAVKMLKENASPSELRDLLSEFNVLKQVNHPHVIKLYGACSQDGPLLLIVEYAKYGSLRGFLRESRKVGPGYLGSGGSRNSSSLDHPDERALTMGDLISFAWQISQGMQYLAEMKLVHRDLAARNILVAEGRKMKISDFGLSRDVYEEDSYVKRSQGRIPVKWMAIESLFDHIYTTQSDVWSFGVLLWEIVTLGGNPYPGIPPERLFNLLKTGHRMERPDNCSEEMYRLMLQCWKQEPDKRPVFADISKDLEKMMVKRRDYLDLAASTPSDSLIYDDGLSEEETPLVDCNNAPLPRALPSTWIENKLYGRISHAFTRF

Accordingly, “RET” or a “RET protein” as used herein encompasses humanRET and variants thereof, including but not limited to orthologsthereof, such as murine RET, cyno RET, and the like. In certainembodiments, RET may be a protein having the amino acid sequence that isat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identicalto SEQ ID NO: 1813.

In certain embodiments, an isolated RET-extracellular domain (RET-ECD)polypeptide is provided. A RET-ECD may be bound to a ligand such as aGFRAL proteion when present in an isolated protein complex of thepresent disclosure. The term “RET-extracellular domain” (“RET-ECD”)includes full-length RET ECDs, RET ECD fragments, and RET ECD variants.As used herein, the term “RET ECD” refers to a RET polypeptide with orwithout a signal peptide that lacks the intracellular and transmembranedomains. In some embodiments, a RET ECD refers to a protein having anamino acid sequence that is at least 75% identical to the amino acidsequence of human full-length RET ECD having the amino acid sequence:

(SEQ ID NO: 1814) KVALGLYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPEDIQDPLCDELCR

In another exemplary embodiment, the a RET ECD refers to a proteinhaving a amino acid sequence that is at least 75% identical to the aminoacid sequence of a human full-length RET ECD having the amino acidsequence:

(SEQ ID NO: 1815) LYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFP EEEKCFCEPEDIQDPLCDELCR

The term “full-length RET ECD”, as used herein, refers to a RET ECD thatextends to the last amino acid of an extracellular domain, and may ormay not include an N-terminal signal peptide. However, it is noted that“full-length RET ECD” also encompasses a RET-ECD that is extended by 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the C-terminus to includeamino acids residues of the transmembrane domain provided that thepolypeptide is soluble. In other words, a RET ECD lacks a sufficientlength of a transmembrane domain such that it is not anchored into acell membrane. The phrase “full-length RET ECD” also encompasses aRET-ECD that is extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidson the N-terminus to include amino acids residues of the signal peptide.In certain embodiments, RET fragment refers to a contiguous amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical to a contiguous amino acid sequence of RETdescribed herein and lacks at least 30, 33, 35, 40, 45, 50, or 55 aminoacids or more at the C-terminus of RET sequences described herein.

As used herein, the term “RET ECD fragment” refers to a RET ECD havingone or more residues deleted from the N and/or C terminus of afull-length ECD and that retains the ability to bind to a GFRAL protein.In some instances, a RET ECD fragment may or may not include anN-terminal signal peptide. In some instances, a RET ECD fragment is ahuman RET ECD fragment that lacks 1, 5, 10, 15, 16, 17, 18, or 19residues present at the N-terminus of the sequence:

(SEQ ID NO: 1816) LYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFP EEEKCFCEPEDIQDPLCDELCR

The above exemplary RET ECD fragment was used in various methodsdescribed in the Examples, including to produce a model of a complexcomprising a RET protein, a GFRAL protein and a GDF15 protein.

In alternative embodiments of a RET ECD, the RET-ECD comprises a C64R,N75Q, N166Q, or C183S mutation in a RET ECD sequence of humanfull-length RET ECD SEQ ID NO 1814.

The phrase “modulates the activity and/or signaling, ” when applied to abinding protein, such an antibody that binds to GFRAL of the presentdisclosure, means that the binding protein (e.g., antibody) mimics ormodulates an in vitro or an in vivo biological effect induced by thebinding of: (i) a GFRAL protein; (ii) a GDF15 protein and a GFRALprotein; or (iii) a GDF15 protein, a GFRAL protein, and a RET protein.In assessing the binding and specificity of anti-GFRAL antibody, forexample, an antibody or fragment thereof, that binds to a GFRAL protein(e.g., a human GFRAL protein), is deemed to induce a biological responsewhen the response is equal to or less than 95%, and preferably equal toor less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95%, of the activity of a wild type GFRALstandard (e.g., the mature form of a human GFRAL protein). An antibodyor fragment thereof, that binds to GFRAL (e.g., human GFRAL), is alsodeemed to induce a biological response when it has one or more of thefollowing properties: exhibiting an efficacy level of equal to or lessthan 95% of a GFRAL standard, with an IC50 of equal to or less than 100nM, e.g., 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM,1 nM, 0.1 nM 0.01 nM in (1) a ELK1-luciferase reporter assay (see, e.g.,Example 3); or (2) ERK-phosphorylation assay in U2OS cells (see, e.g.,Example 4).

The term “binding protein” refers to a protein comprising a portion(e.g., one or more binding regions such as CDRs) that binds to a GFRALprotein, including a human GFRAL protein and, optionally, a scaffold orframework portion (e.g., one or more scaffold or framework regions) thatallows the binding portion to adopt a conformation that promotes bindingof the binding protein to a GFRAL polypeptide, fragment or epitope.Examples of such binding proteins include antibodies, such as a humanantibody, a humanized antibody; a chimeric antibody; a recombinantantibody; a single chain antibody; a diabody; a triabody; a tetrabody; aFab fragment; a F(ab′) 2 fragment; an IgD antibody; an IgE antibody; anIgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; oran IgG4 antibody, and fragments thereof. The binding protein cancomprise, for example, an alternative protein scaffold or artificialscaffold with grafted CDRs or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe binding protein as well as wholly synthetic scaffolds comprising,for example, a biocompatible polymer. See, e.g., K_(D) rndorfer et al.,2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129(2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition,peptide antibody mimetics (“PAMs”) can be used, as well as scaffoldsbased on antibody mimetics utilizing fibronectin components as ascaffold. In the context of the present disclosure, a binding protein issaid to specifically bind or selectively bind to GFRAL, for example,when the dissociation constant (K_(D)) is ≤10⁻⁸ M. The binding protein(e.g.,antibody) may specifically bind GFRAL with high affinity when theK_(D) is ≤10⁻⁹ M or K_(D) is ≤10⁻¹⁰ M. In some embodiments, the bindingproteins (e.g., antibodies) may bind to GFRAL, including with a K_(D) ofbetween about 10⁻⁷ M and about 10⁻¹² M and in other embodiments, thebinding proteins (e.g., antibodies) may bind with a K_(D) of 1−2×10⁻⁹ M.

The term “antibody” and “immunoglobulin” or “Ig” are usedinterchangeably herein, and is used in the broadest sense andspecifically covers, for example, individual anti-GFRAL monoclonalantibodies (including agonist, antagonist, neutralizing antibodies, fulllength or intact monoclonal antibodies), anti-GFRAL antibodycompositions with polyepitopic or monoepitopic specificity, polyclonalor monovalent antibodies, multivalent antibodies, multispecificantibodies (e.g., bispecific antibodies so long as they exhibit thedesired biological activity), formed from at least two intactantibodies, single chain anti-GFRAL antibodies, and fragments ofanti-GFRAL antibodies, as described below. An antibody can be human,humanized, chimeric and/or affinity matured as well as an antibody fromother species, for example mouse, rabbit etc. The term “antibody” isintended to include a polypeptide product of B cells within theimmunoglobulin class of polypeptides that is able to bind to a specificmolecular antigen and is composed of two identical pairs of polypeptidechains, wherein each pair has one heavy chain (about 50-70 kDa) and onelight chain (about 25 kDa) and each amino-terminal portion of each chainincludes a variable region of about 100 to about 130 or more amino acidsand each carboxy-terminal portion of each chain includes a constantregion (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed.,Oxford University Press.; Kuby (1997) Immunology, Third Ed., W. H.Freeman and Company, N.Y.). In specific embodiments, the specificmolecular antigen can be bound by an antibody provided herein includes aGFRAL polypeptide, GFRAL fragment or GFRAL epitope. Antibodies alsoinclude, but are not limited to, synthetic antibodies, monoclonalantibodies, recombinantly produced antibodies, multispecific antibodies(including bi-specific antibodies), human antibodies, humanizedantibodies, camelized antibodies, chimeric antibodies, intrabodies,anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g.,antigens-binding fragments such as GFRAL binding fragments) of any ofthe above, which refers a portion of an antibody heavy or light chainpolypeptide that retains some or all of the binding activity of theantibody from which the fragment was derived. Non-limiting examples offunctional fragments (e.g., antigens-binding fragments such as GFRALbinding fragments) include single-chain Fvs (scFv) (e.g., includingmonospecific, bispecific, etc.), Fab fragments, F(ab′) fragments, F(ab)2fragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments,Fv fragments, diabody, triabody, tetrabody and minibody. In particular,antibodies provided herein include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, forexample, antigen binding domains or molecules that contain anantigen-binding site that binds to a GFRAL antigen (e.g., one or morecomplementarity determining regions (CDRs) of an anti-GFRAL antibody).Such antibody fragments can be found described in, for example, Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1989); Myers (ed.), Molec. Biology and Biotechnology:A Comprehensive Desk Reference, N.Y.: VCH Publisher, Inc.; Huston etal., Cell Biophysics, 22:189-224 (1993); PlUckthun and Skerra, Meth.Enzymol., 178:497-515 (1989) and in Day, E. D., AdvancedImmunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990).The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulinmolecule. Anti-GFRAL antibodies may be agonistic antibodies orantagonistic antibodies. Antibodies provided herein include antagonisticantibodies to GFRAL, for example, antibodies that inhibit GFRALsignaling. Exemplary anti-GFRAL antibodies include antibodies with CDRsas shown in Tables 1-24.

The terms “about” or “approximately” mean within 20%, within 15%, within10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%,within 3%, within 2%, within or 1% or less of a given value or range.

An “antigen” is a predetermined antigen to which an antibody canselectively bind. A target antigen may be a polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. In some embodiments, the target antigen is a polypeptide.

The term “antigen binding fragment,” “antigen binding domain,” “antigenbinding region,” and similar terms refer to that portion of an antibodywhich comprises the amino acid residues that interact with an antigenand confer on the binding agent its specificity and affinity for theantigen (e.g., the complementarity determining regions (CDRs)).

The terms “binds” or “binding” refer to an interaction (e.g., covalentor non-covalent) between molecules including, for example, to form acomplex. A complex can also include the binding of two or more moleculesheld together by covalent or non-covalent bonds, interactions or forces.Interactions can be, for example, non-covalent interactions includinghydrogen bonds, ionic bonds, hydrophobic interactions, and/or van derWaals interactions. The strength of the total non-covalent interactionsbetween a single antigen-binding site on an antibody and a singleepitope of a target molecule, such as GFRAL, is the affinity of theantibody or functional fragment for that epitope. The ratio ofassociation (k1) to dissociation (k−1) of an antibody to a monovalentantigen (k1/k−1) is the association constant K, which is a measure ofaffinity. The value of K varies for different complexes of antibody andantigen and depends on both k1 and k−1. The association constant K foran antibody provided herein can be determined using any method providedherein or any other method well known to those skilled in the art. Theaffinity at one binding site does not always reflect the true strengthof the interaction between an antibody and an antigen. When complexantigens containing multiple, repeating antigenic determinants, such asa polyvalent GFRAL, come in contact with antibodies containing multiplebinding sites, the interaction of antibody with antigen at one site willincrease the probability of a reaction at a second site. The strength ofsuch multiple interactions between a multivalent antibody and antigen iscalled the avidity. The avidity of an antibody can be a better measureof its binding capacity than is the affinity of its individual bindingsites. For example, high avidity can compensate for low affinity as issometimes found for pentameric IgM antibodies, which can have a loweraffinity than IgG, but the high avidity of IgM, resulting from itsmultivalence, enables it to bind antigen effectively.

The terms “antibodies that specifically bind to GFRAL,” “antibodies thatspecifically bind to a GFRAL epitope,” and analogous terms are also usedinterchangeably herein and refer to antibodies that specifically bind toa GFRAL polypeptide, such as a GFRAL antigen, or fragment, or epitope(e.g., human GFRAL such as a human GFRAL polypeptide, antigen orepitope). An antibody that specifically binds to GFRAL, (e.g., humanGFRAL) may bind to the extracellular domain or peptide derived from theextracellular domain of GFRAL. An antibody that specifically binds to aGFRAL antigen (e.g., human GFRAL) may be cross-reactive with relatedantigens (e.g., cyno GFRAL). In certain embodiments, an antibody thatspecifically binds to a GFRAL antigen does not cross-react with otherantigens. An antibody that specifically binds to a GFRAL antigen can beidentified, for example, by immunoassays, Biacore, or other techniquesknown to those of skill in the art. An antibody binds specifically to aGFRAL antigen when it binds to a GFRAL antigen with higher affinity thanto any cross reactive antigen as determined using experimentaltechniques, such as radioimmunoassays (RIA) and enzyme linkedimmunosorbent assays (ELISAs). Typically a specific or selectivereaction will be at least twice background signal or noise and may bemore than 10 times background. See, e.g., Paul, ed., 1989, FundamentalImmunology Second Edition, Raven Press, N.Y. at pages 332 336 for adiscussion regarding antibody specificity. An antibody “which binds” anantigen of interest (e.g., a target antigen such as GFRAL) is one thatbinds the antigen with sufficient affinity such that the antibody isuseful as a therapeutic agent in targeting a cell or tissue expressingthe antigen, and does not significantly cross-react with other proteins.In such embodiments, the extent of binding of the antibody to a“non-target” protein will be less than about 10% of the binding of theantibody to its particular target protein, for example, as determined byfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA). With regard to the binding of anantibody to a target molecule, the term “specific binding” or“specifically binds to” or is “specific for” a particular polypeptide oran epitope on a particular polypeptide target means binding that ismeasurably different from a non-specific interaction. Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a moleculeof similar structure that does not have binding activity. For example,specific binding can be determined by competition with a controlmolecule that is similar to the target, for example, an excess ofnon-labeled target. In this case, specific binding is indicated if thebinding of the labeled target to a probe is competitively inhibited byexcess unlabeled target. The term “specific binding” or “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide target as used herein can be exhibited, forexample, by a molecule having a Kd for the target of at least about10⁻⁴M, alternatively at least about 10⁻⁵ M, alternatively at least about10⁻⁶ M, alternatively at least about 10⁻⁷ M, alternatively at leastabout 10⁻⁸ M, alternatively at least about 10⁻⁹ M, alternatively atleast about 10⁻¹⁰ M, alternatively at least about 10⁻¹¹ M, alternativelyat least about 10⁻¹² M, or greater. In one embodiment, the term“specific binding” refers to binding where a molecule binds to aparticular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.In certain embodiments, an antibody that binds to GFRAL has adissociation constant (Kd) of less than or equal to 10 nM, 5 nM, 4 nM, 3nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM,0.2 nM, or 0.1 nM. The lower the K_(D) , the higher the affinity of theanti-GFRAL antibody. In certain embodiments, anti- GFRAL antibody bindsto an epitope of GFRAL that is conserved among GFRAL from differentspecies (e.g., between human and cyno GFRAL).

The term “compete” when used in the context of anti-GFRAL antibodies(e.g., antagonistic antibodies and binding proteins that bind to GFRAL)that bind to or compete for the same epitope or binding site on a targetmeans competition between as determined by an assay in which theantibody (or binding fragment) thereof under study prevents or inhibitsthe specific binding of a reference molecule (e.g., a reference ligand,or reference antigen binding protein, such as a reference antibody) to acommon antigen (e.g., GFRAL or a fragment thereof). Numerous types ofcompetitive binding assays can be used to determine if a test antibodycompetes with a reference antibody for binding to GFRAL (e.g., humanGFRAL). Examples of assays that can be employed include solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (see,e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solidphase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J.Immunol. 137:3614-3619) solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see, e.g., Harlow and Lane, (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label RIA using 1-125 label (see, e.g., Morel et al., (1988)Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see,e.g., Cheung, et al., (1990) Virology 176:546-552); and direct labeledRIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically,such an assay involves the use of a purified antigen (e.g., GFRAL suchas human GFRAL) bound to a solid surface or cells bearing either of anunlabelled test antigen binding protein (e.g., test anti-GFRAL antibody)or a labeled reference antigen binding protein (e.g., referenceanti-GFRAL antibody). Competitive inhibition may be measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test antigen binding protein. Usually the testantigen binding protein is present in excess. Antibodies identified bycompetition assay (competing antibodies) include antibodies binding tothe same epitope as the reference antibody and/or antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference for antibodies steric hindrance to occur. Additional detailsregarding methods for determining competitive binding and/or binding tothe same epitope are described herein. Usually, when a competingantibodies protein is present in excess, it will inhibit specificbinding of a reference antibodies to a common antigen by at least 23%,for example 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance,binding is inhibited by at least 80%, 85%, 90%, 95%, 96% or 97%, 98%,99% or more.

The term “anti-GFRAL antibody” or “an antibody that binds to GFRAL”includes an antibody that is capable of binding GFRAL with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting GFRAL. In certain embodiments, the extentof binding of an anti-GFRAL antibody to an unrelated, non-GFRAL proteinis less than about 10% of the binding of the antibody to GFRAL asmeasured, for example, by fluorescence activated cell sorting (FACS)analysis or an immunoassay such as a radioimmunoassay (RIA). An antibodythat “specifically binds to” or is “specific for” GFRAL is illustratedherein. In certain embodiments, an antibody that binds to GFRAL, asdescribed herein, has a dissociation constant (Kd) of less than or equalto 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 0.9 nM, 0.8 nM, 0.7 nM,0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM, and/or is greaterthan or equal to 0.1nM. In certain embodiments, anti-GFRAL antibodybinds to an epitope of GFRAL that is conserved among GFRAL fromdifferent species (e.g., between human and cyno GFRAL).

The terms “crystal”, and “crystallized” as used herein, refer to one ormore proteins or fragments thereof that exist in the form of a crystal.Crystals are one form of the solid state of matter, which is distinctfrom other forms such as the amorphous solid state or the liquidcrystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., ligand/receptor orantigen/antibody complexes). These three-dimensional arrays are arrangedaccording to specific mathematical relationships that arewell-understood in the field. The fundamental unit, or building block,that is repeated in a crystal is called the asymmetric unit. Repetitionof the asymmetric unit in an arrangement that conforms to a given,well-defined crystallographic symmetry provides the “unit cell” of thecrystal. Repetition of the unit cell by regular translations in allthree dimensions provides the crystal. See Giege, R. and Ducruix, A.Barrett, Crystallization of Nucleic Acids and Proteins, a PracticalApproach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y.,(1999).

An “isolated” antibody is substantially free of cellular material orother contaminating proteins from the cell or tissue source and/or othercontaminant components from which the antibody is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of an antibody in which the antibody isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, an antibody that issubstantially free of cellular material includes preparations ofantibody having less than about 30%, 25%, 20%, 15%,10%, 5%, or 1% (bydry weight) of heterologous protein (also referred to herein as a“contaminating protein”). In certain embodiments, when the antibody isrecombinantly produced, it is substantially free of culture medium,e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1%of the volume of the protein preparation. In certain embodiments, whenthe antibody is produced by chemical synthesis, it is substantially freeof chemical precursors or other chemicals, for example, it is separatedfrom chemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the antibodyhave less than about 30%, 25%, 20%, 15%, 10%, 5% or 1% (by dry weight)of chemical precursors or compounds other than the antibody of interest.Contaminant components can also include, but are not limited to,materials that would interfere with therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method (Lowry et al. J. Bio. Chem. 193: 265-275, 1951), suchas 96%, 97%, 98%, or 99%, by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step. Inspecific embodiments, antibodies provided herein are isolated.

A 4-chain antibody unit is a heterotetrameric glycoprotein composed oftwo identical light (L) chains and two identical heavy (H) chains. Inthe case of IgGs, the 4-chain unit is generally about 150,000 daltons.Each L chain is linked to a H chain by one covalent disulfide bond,while the two H chains are linked to each other by one or more disulfidebonds depending on the H chain isotype. Each H and L chain also hasregularly spaced intrachain disulfide bridges. Each H chain has at theN-terminus, a variable domain (VH) followed by three constant domains(CH) for each of the α and γ chains and four CH domains for μ and ϵisotypes. Each L chain has at the N-terminus, a variable domain (VL)followed by a constant domain (CL) at its other end. The VL is alignedwith the VH and the CL is aligned with the first constant domain of theheavy chain (CH1). Particular amino acid residues are believed to forman interface between the light chain and heavy chain variable domains.The pairing of a VH and VL together forms a single antigen-binding site.For the structure and properties of the different classes of antibodies,see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites,Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk,Conn., 1994, page 71 and Chapter 6.

The term “variable region” or “variable domain” refers to a portion ofthe light or heavy chains of an antibody that is generally located atthe amino-terminal of the light or heavy chain and has a length of about120 to 130 amino acids in the heavy chain and about 100 to 110 aminoacids in the light chain, and are used in the binding and specificity ofeach particular antibody for its particular antigen. The variable regionof the heavy chain may be referred to as “VH.” The variable region ofthe light chain may be referred to as “VL.” The term “variable” refersto the fact that certain segments of the variable regions differextensively in sequence among antibodies. The V region mediates antigenbinding and defines specificity of a particular antibody for itsparticular antigen. However, the variability is not evenly distributedacross the 110-amino acid span of the variable regions. Instead, the Vregions consist of less variable (e.g., relatively invariant) stretchescalled framework regions (FRs) of about 15-30 amino acids separated byshorter regions of greater variability (e.g., extreme variability)called “hypervariable regions” that are each about 9-12 amino acidslong. The variable regions of heavy and light chains each comprise fourFRs, largely adopting a β sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the β sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991)). Theconstant regions are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody dependent cellular cytotoxicity (ADCC) andcomplement dependent cytotoxicity (CDC). The variable regions differextensively in sequence between different antibodies. The variability insequence is concentrated in the CDRs while the less variable portions inthe variable region are referred to as framework regions (FR). The CDRsof the light and heavy chains are primarily responsible for theinteraction of the antibody with antigen. In specific embodiments, thevariable region is a human variable region.

The term “variable region residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable regions or light chainvariable regions of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc, according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. The Kabatnumbering system is generally used when referring to a residue in thevariable domain (approximately residues 1-107 of the light chain andresidues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG 1 EU antibody. Other numberingsystems have been described, including, for example, by AbM, Chothia,Contact, IMGT and AHon. Various numbering systems are illustrated inTables 1-24.

An “intact” antibody is one comprising an antigen-binding site as wellas a light chain constant region CL and at least heavy chain constantregions, CH1, CH2 and CH3. The constant regions may include humanconstant regions or amino acid sequence variants thereof. Preferably, anintact antibody has one or more effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include, without limitation,Fab, Fab′, F(ab′)2, and Fv fragments; diabodies and di-diabodies (see,e.g., Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. 90:6444-8; Lu,D. et al., (2005) J. Biol. Chem. 280:19665-72; Hudson et al., Nat. Med.9:129-134 (2003); WO 93/11161; and U.S. Pat. Nos. 5,837,242 and6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos.4,946,778; 5,260,203; 5,482,858 and 5,476,786); dual variable domainantibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domainantibodies (SdAbs) (see, e.g., Woolven et al., Immunogenetics 50:98-101, 1999; Streltsov et al., Proc Natl Acad Sci USA. 101:12444-12449,2004); and multispecific antibodies formed from antibody fragments.

A “functional fragment” or “binding fragment” or “antigen bindingfragment” of a therapeutic antibody will exhibit at least one if notsome or all of the biological functions attributed to the intactantibody, the function comprising at least binding to the targetantigen, (e.g., a GFRAL binding fragment or fragment that binds toGFRAL).

The term “fusion protein” as used herein refers to a polypeptide thatcomprises an amino acid sequence of an antibody and an amino acidsequence of a heterologous polypeptide or protein (e.g., a polypeptideor protein not normally a part of the antibody (e.g., a non-anti-GFRALantigen binding antibody)). The term “fusion” when used in relation toGFRAL or to an anti-GFRAL antibody refers to the joining of a peptide orpolypeptide, or fragment, variant and/or derivative thereof, with aheterologous peptide or polypeptide. In certain embodiments, the fusionprotein retains the biological activity of the GFRAL or anti-GFRALantibody. In certain embodiments, the fusion protein comprises a GFRALantibody VH region, VL region, VH CDR (one, two or three VH CDRs),and/or VL CDR (one, two or three VL CDRs), wherein the fusion proteinbinds to a GFRAL epitope, a GFRAL fragment and/or a GFRAL polypeptide.

The term “heavy chain” when used in reference to an antibody refers to apolypeptide chain of about 50-70 kDa, wherein the amino-terminal portionincludes a variable region of about 120 to 130 or more amino acids and acarboxy-terminal portion that includes a constant region (e.g., CH1,CH2, CH3, CH4). The constant region can be one of five distinct types,(e.g., isotypes) referred to as alpha (α), delta (δ), epsilon (ϵ), gamma(γ) and mu (μ), based on the amino acid sequence of the heavy chainconstant regions. The distinct heavy chains differ in size: α, δ and γcontain approximately 450 amino acids, while p and c containapproximately 550 amino acids. When combined with a light chain, thesedistinct types of heavy chains give rise to five well known classes(e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG and IgM,respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to apolypeptide chain of about 25 kDa, wherein the amino-terminal portionincludes a variable region of about 100 to about 110 or more amino acidsand a carboxy-terminal portion that includes a constant region. Theapproximate length of a light chain is 211 to 217 amino acids. There aretwo distinct types, referred to as kappa (κ) of lambda (λ) based on theamino acid sequence of the constant domains. Light chain amino acidsequences are well known in the art. A light chain can be a human lightchain.

The term “effective amount” as used herein refers to the amount of atherapy (e.g., an anti-GFRAL antibody or pharmaceutical compositionprovided herein; see, e.g., antibodies comprising CDR, VH, and/or VLsequences as shown in Tables 1-24) which is sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with a GDF15-mediated disease, disorder, orcondition, including, for example, involuntary body weight loss, aglucose metabolism disorder or a body weight disorder. This term alsoencompasses an amount necessary for the reduction or amelioration of theadvancement or progression of a given GDF15-mediated disease, disorderor condition, reduction or amelioration of the recurrence, developmentor onset of a GDF15-mediated disease, disorder or condition, and/or toimprove or enhance the prophylactic or therapeutic effect(s) of anothertherapy (e.g., a therapy other than anti-GFRAL antibody providedherein). In some embodiments, the effective amount is administered inone or more doses, including intermittent doses, wherein one ore moredoses are given in a treatment period followed by a resting period whenan antibody is not administered (e.g., one cycle of treatment period andrest period can be followed with additional cycles, with one or moretreatment periods followed by one or more resting periods). In someembodiments, the effective amount of an antibody provided herein is fromabout 0.1 mg/kg (mg of antibody per kg weight of the subject) to about100 mg/kg. In certain embodiments, an effective amount of an antibodyprovided therein is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3mg/kg, 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg,about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90mg/kg, or about 100 mg/kg (or a range therein). In some embodiments,effective amount as used herein also refers to the amount of an antibodyprovided herein to achieve a specified result (e.g., mimic or modulatean in vitro or an in vivo biological effect induced by the binding of:(i) GFRAL; (ii) GDF15 and GFRAL; or (iii) GDF15, GFRAL, and RET). Forexample, an effective amount includes an amount (e.g., in one or moredoses) of an anti-GFRAL antibody as described herein effective to: (i)increase body weight; (ii) maintain body weight; (iii) reduce bodyweight loss; (iv) increase body mass (e.g., lean mass or fat mass); (v)maintain body mass (e.g., lean mass or fat mass); or (vi) reduce loss ofbody mass (e.g., lean mass or fat mass).

The term “host cell” as used herein refers to a particular subject cellthat may be transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transfected with the nucleic acid moleculedue to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome.

The term “immunomodulatory agent” and variations thereof including, butnot limited to, immunomodulatory agents, as used herein refer to anagent that modulates a host's immune system. In certain embodiments, animmunomodulatory agent used in the combination therapies provided hereindoes not include an anti-GFRAL antibody or antigen-binding fragment.Immunomodulatory agents include, but are not limited to, smallmolecules, peptides, polypeptides, proteins, fusion proteins,antibodies, inorganic molecules, mimetic agents, and organic molecules.The term “small molecule” and analogous terms include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acidanalogues, polynucleotides, polynucleotide analogues, nucleotides,nucleotide analogues, organic or inorganic compounds (i.e., includingheterorganic and/or ganometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds. In someembodiments, an immunomodulatory agent is an immunostimulatory agent. Insome embodiments, an immunomodulatory agent is an immunosuppressantagent. In some embodiments, immunomodulatory agents are agents (e.g.,antibodies) that modulate (e.g., inhibit or stimulate) proteins known asimmune checkpoint molecules (e.g., co-inhibitory or co-stimulatory), forexample, C10orf54, CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2,B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160,BTLA, HVEM, LAIR1, TIM1, Galectin 9, TIM3, CD48, 2B4, CD155, CD112,CD113 and TIGIT (e.g., co-inhibitory), and/or CD154, TNFRSF25, GITR,4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L,CD70, HHLA2, ICOSL, a cytokine, LIGHT, HVEM, CD30, CD3OL, B7-H2, CD80,CD86, CD4OL, TIM4, TIM1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2,and CD226 (e.g., co-stimulatory).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts, and each monoclonal antibody will typically recognize asingle epitope on the antigen. In specific embodiments, a “monoclonalantibody,” as used herein, is an antibody produced by a single hybridomaor other cell, wherein the antibody binds to only a GFRAL epitope asdetermined, for example, by ELISA or other antigen-binding orcompetitive binding assay known in the art. The term “monoclonal” is notlimited to any particular method for making the antibody. For example,the monoclonal antibodies useful in the present disclosure may beprepared by the hybridoma methodology first described by Kohler et al.,Nature, 256:495 (1975), or may be made using recombinant DNA methods inbacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991), for example. Other methods for the preparation of clonal celllines and of monoclonal antibodies expressed thereby are well known inthe art (see, for example, Chapter 11 in: Short Protocols in MolecularBiology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons,N.Y.). Exemplary methods of producing monoclonal antibodies are providedin the Examples herein.

The term “native” when used in connection with biological materials suchas nucleic acid molecules, polypeptides, host cells, and the like,refers to those which are found in nature and not manipulated, modified,and/or changed (e.g., isolated, purified, selected) by a human being.

The antibodies provided herein can include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of nonhuman (e.g., murine) antibodies are chimericantibodies that include human immunoglobulins (e.g., recipient antibody)in which the native CDR residues are replaced by residues from thecorresponding CDR of a nonhuman species (e.g., donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, one or more FR regionresidues of the human immunoglobulin are replaced by correspondingnonhuman residues. Furthermore, humanized antibodies can compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. A humanized antibody heavy or light chain can comprisesubstantially all of at least one or more variable regions, in which allor substantially all of the CDRs correspond to those of a nonhumanimmunoglobulin and all or substantially all of the FRs are those of ahuman immunoglobulin sequence. In certain embodiments, the humanizedantibody will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. For furtherdetails, see, Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992); Carter et al., Proc. Natl. Acd. Sci. USA 89:4285-4289(1992); and U.S. Pat. No. 6,800,738 (issued Oct. 5, 2004), U.S. Pat. No.6,719,971 (issued Sept. 27, 2005), U.S. Pat. No. 6,639,055 (issued Oct.28, 2003), U.S. Pat. No. 6,407,213 (issued Jun. 18, 2002), and U.S. Pat.No. 6,054,297 (issued Apr. 25, 2000).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries (Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991) and yeast display libraries (Chao et al., NatureProtocols 1: 755-768 (2006)). Also available for the preparation ofhuman monoclonal antibodies are methods described in Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk andvan de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Humanantibodies can be prepared by administering the antigen to a transgenicanimal that has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled, e.g.,mice (see, e.g., Jakobovits, A., Curr. Opin. Biotechnol. 1995,6(5):561-6; BrUggemann and Taussing, Curr. Opin. Biotechnol. 1997,8(4):455-8; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

A “CDR” refers to one of three hypervariable regions (H1, H2 or H3)within the non-framework region of the immunoglobulin (Ig or antibody)VH β-sheet framework, or one of three hypervariable regions (L1, L2 orL3) within the non-framework region of the antibody VL β-sheetframework. Accordingly, CDRs are variable region sequences interspersedwithin the framework region sequences. CDR regions are well known tothose skilled in the art and have been defined by, for example, Kabat asthe regions of most hypervariability within the antibody variable (V)domains (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv.Prot. Chem. 32:1-75 (1978)). CDR region sequences also have been definedstructurally by Chothia as those residues that are not part of theconserved β-sheet framework, and thus are able to adapt differentconformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Bothterminologies are well recognized in the art. CDR region sequences havealso been defined by AbM, Contact and IMGT. CDR region sequences areillustrated in Tables 1-24. The positions of CDRs within a canonicalantibody variable region have been determined by comparison of numerousstructures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Moreaet al., Methods 20:267-279 (2000)). Because the number of residueswithin a hypervariable region varies in different antibodies, additionalresidues relative to the canonical positions are conventionally numberedwith a, b, c and so forth next to the residue number in the canonicalvariable region numbering scheme (Al-Lazikani et al., supra (1997)).Such nomenclature is similarly well known to those skilled in the art.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable region that are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (see, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)). Chothiarefers instead to the location of the structural loops (see,e.g.,Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of theChothia CDR-H1 loop when numbered using the Kabat numbering conventionvaries between H32 and H34 depending on the length of the loop (this isbecause the Kabat numbering scheme places the insertions at H35A andH35B; if neither 35A nor 35B is present, the loop ends at 32; if only35A is present, the loop ends at 33; if both 35A and 35B are present,the loop ends at 34). The AbM hypervariable regions represent acompromise between the Kabat CDRs and Chothia structural loops, and areused by Oxford Molecular's AbM antibody modeling software (see, e.g.,Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag).The “contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions or CDRs are noted below.

Recently, a universal numbering system has been developed and widelyadopted, ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev.Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated informationsystem specializing in immunoglobulins (IG), T cell receptors (TR) andmajor histocompatibility complex (MHC) of human and other vertebrates.Herein, the CDRs are referred to in terms of both the amino acidsequence and the location within the light or heavy chain. As the“location” of the CDRs within the structure of the immunoglobulinvariable domain is conserved between species and present in structurescalled loops, by using numbering systems that align variable domainsequences according to structural features, CDR and framework residuesand are readily identified. This information can be used in grafting andreplacement of CDR residues from immunoglobulins of one species into anacceptor framework from, typically, a human antibody. An additionalnumbering system (AHon) has been developed by Honegger and Plückthun, J.Mol. Biol. 309: 657-670 (2001). Correspondence between the numberingsystem, including, for example, the Kabat numbering and the IMGT uniquenumbering system, is well known to one skilled in the art (see, e.g.,Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al.,supra) and is also illustrated in Tables 1-24. An Exemplary system,shown herein, combines Kabat and Chothia.

Exemplary IMGT Kabat AbM Chothia Contact V_(H) CDR1 26-35  27-38 31-3526-35 26-32 30-35 V_(H) CDR2 50-65  56-65 50-65 50-58 53-55 47-58 V_(H)CDR3 95-102 105-117 95-102 95-102 96-101 93-101 V_(L) CDR1 24-34  27-3824-34 24-34 26-32 30-36 V_(L) CDR2 50-56  56-65 50-56 50-56 50-52 46-55V_(L) CDR3 89-97 105-117 89-97 89-97 91-96 89-96

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96(L3) in the VL and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102,94-102, or 95-102 (H3) in the VH. As used herein, the terms “HVR” and“CDR” are used interchangeably.

The term “constant region” or “constant domain” refers to a carboxyterminal portion of the light and heavy chain which is not directlyinvolved in binding of the antibody to antigen but exhibits variouseffector function, such as interaction with the Fc receptor. The termsrefer to the portion of an immunoglobulin molecule having a moreconserved amino acid sequence relative to the other portion of theimmunoglobulin, the variable region, which contains the antigen bindingsite. The constant region may contain the CH1, CH2 and CH3 regions ofthe heavy chain and the CL region of the light chain.

The term “framework” or “FR” residues are those variable region residuesflanking the CDRs. FR residues are present, for example, in chimeric,humanized, human, domain antibodies, diabodies, linear antibodies, andbispecific antibodies. FR residues are those variable domain residuesother than the hypervariable region residues or CDR residues.

An “affinity matured” antibody is one with one or more alterations(e.g., amino acid sequence variations, including changes, additionsand/or deletions) in one or more HVRs thereof which result in animprovement in the affinity of the antibody for antigen, compared to aparent antibody which does not possess those alteration(s). Preferredaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity matured antibodies areproduced by procedures known in the art. For review, see Hudson andSouriau, Nature Medicine 9 :129-134 (2003); Hoogenboom, NatureBiotechnol. 23 : 1105-1116 (2005); Quiroz and Sinclair, RevistaIngeneria Biomedia 4 : 39-51 (2010).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds (e.g., GFRAL, asdescribed herein). For example, blocking antibodies or antagonistantibodies may substantially or completely inhibit the biologicalactivity of the antigen (e.g., GFRAL, as described herein).

An “agonist antibody” is an antibody that triggers a response, e.g., onethat mimics at least one of the functional activities of a polypeptideof interest. An agonist antibody includes an antibody that is a ligandmimetic, for example, wherein a ligand binds to a cell surface receptorand the binding induces cell signaling or activities via anintercellular cell signaling pathway and wherein the antibody induces asimilar cell signaling or activation.

An “antagonist” of GFRAL refers to a molecule (e.g., antibody) that iscapable of detectably inhibiting or otherwise decreasing one or more ofthe biological activities of GFRAL, such as in a cell expressing GFRAL.In some embodiments, an antagonist of GFRAL (e.g., an agonistic antibodyas described herein) may, for example, act by detectably inhibiting orotherwise decreasing the activation and/or cell signaling pathways of acell expressing a GFRAL, thereby detectably decreasing a GFRAL-mediatedbiological activity of the cell relative to the GFRAL-mediatedbiological activity in the absence of antagonist. In some embodimentsthe antibodies provided herein are antagonistic anti-GFRAL antibodies,including antibodies that inhibit signaling of a complex comprisingGFRAL, GDF15 and/or RET. The inhibition or decrease caused by a GFRALantagonist need not be complete as long as it is detectable using anassay. For example, a cell-based assay described in the Examples belowcan be used to analyze a biological activity of GFRAL.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., a binding protein such as an antibody) and its binding partner(e.g., an antigen). Unless indicated otherwise, as used herein, “bindingaffinity” refers to intrinsic binding affinity which reflects a 1:1interaction between members of a binding pair (e.g., antibody andantigen). The affinity of a binding molecule X for its binding partner Ycan generally be represented by the dissociation constant (K_(D)).Affinity can be measured by common methods known in the art, includingthose described herein. Low-affinity antibodies generally bind antigenslowly and tend to dissociate readily, whereas high-affinity antibodiesgenerally bind antigen faster and tend to remain bound longer. A varietyof methods of measuring binding affinity are known in the art, any ofwhich can be used for purposes of the present disclosure. Specificillustrative embodiments include the following. In one embodiment, the“K_(D)” or “K_(D) value” may be measured by assays known in the art, forexample by a binding assay. The K_(D) may be measured in a radiolabeledantigen binding assay (RIA), for example, performed with the Fab versionof an antibody of interest and its antigen (Chen, et al., (1999) J. MolBiol 293:865-881). The K_(D) or K_(D) value may also be measured byusing surface plasmon resonance assays by Biacore, using, for example, aBIAcore™-2000 or a BIAcore™-3000 BIAcore, Inc., Piscataway, N.J.), or bybiolayer interferometry using, for example, the OctetQK384 sytem(ForteBio, Menlo Park, Calif.). An “on-rate” or “rate of association” or“association rate” or “kon” may can also be determined with the samesurface plasmon resonance or biolayer interferometry techniquesdescribed above using, for example, a BIAcore™-2000 or a BIAcore™-3000(BIAcore, Inc., Piscataway, N.J.), or the OctetQK384 sytem (ForteBio,Menlo Park, Calif.).

The phrase “substantially similar” or “substantially the same” denotes asufficiently high degree of similarity between two numeric values (e.g.,one associated with an antibody of the present disclosure and the otherassociated with a reference antibody) such that one of skill in the artwould consider the difference between the two values to be of little orno biological and/or statistical significance within the context of thebiological characteristic measured by the values (e.g., K_(D) values).For example, the difference between the two values may be less thanabout 50%, less than about 40%, less than about 30%, less than about20%, less than about 10%, less than about 5%,as a function of the valuefor the reference antibody.

The phrase “substantially reduced,” or “substantially different”, asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (e.g., one associated with an antibody of the presentdisclosure and the other associated with a reference antibody) such thatone of skill in the art would consider the difference between the twovalues to be of statistical significance within the context of thebiological characteristic measured by the values. For example, thedifference between said two values may be preferably greater than about10%, greater than about 20%, greater than about 30%, greater than about40%, greater than about 50% as a function of the value for the referenceantibody.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (e.g., a native sequence Fc region oramino acid sequence variant Fc region) of an antibody, and vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including, for example, native sequence Fcregions, recombinant Fc regions, and variant Fc regions. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is often defined to stretch from anamino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The C-terminal lysine (residue 447 accordingto the EU numbering system) of the Fc region may be removed, forexample, during production or purification of the antibody, or byrecombinantly engineering the nucleic acid encoding a heavy chain of theantibody. Accordingly, a composition of intact antibodies may compriseantibody populations with all K447 residues removed, antibodypopulations with no K447 residues removed, and antibody populationshaving a mixture of antibodies with and without the K447 residue.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding region or binding domain (e.g., an antibody variableregion or domain) and can be assessed, including using various assays asdisclosed herein and/or as known in the art.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature, and notmanipulated, modified, and/or changed (e.g., isolated, purified,selected, including or combining with other sequences such as variableregion sequences) by a human. Native sequence human Fc regions include anative sequence human IgG1 Fc region (non-A and A allotypes); nativesequence human IgG2 Fc region; native sequence human IgG3 Fc region; andnative sequence human IgG4 Fc region as well as naturally occurringvariants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, (e.g., substituting, addition, or deletion)preferably one or more amino acid substitution(s). Preferably, thevariant Fc region has at least one amino acid substitution compared to anative sequence Fc region or to the Fc region of a parent polypeptide,for example, from about one to about ten amino acid substitutions, andpreferably from about one to about five amino acid substitutions in anative sequence Fc region or in the Fc region of the parent polypeptide.The variant Fc region herein will preferably possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of aparent polypeptide, and more preferably at least about 90% homologytherewith, for example, at least about 95% homology therewith. Forexample, a variant with two amino acid changes to alanine at twopositions in the human IgG1 Fc sequence are shown bolded in the aminoacid sequence provided below:

(SEQ ID NO: 2001) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

For example, a variant with two amino acid changes to alanine at twopositions in a truncated human IgG1 Fc sequence, in which the C-terminallysine residue is absent (IgG1ΔK Fc), are shown bolded in the amino acidsequence provided below:

(SEQ ID NO: 2002) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

For example, a truncated variant of the human IgG1 Fc sequence, in whichthe C-terminal lysine residue is absent (IgG1ΔK Fc) is shown in theamino acid sequence provided below:

(SEQ ID NO: 2003) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

For example, a variant with an amino acid change to proline at aposition in the human IgG4 Fc sequence is shown bolded in the amino acidsequence provided below:

(SEQ ID NO: 2004) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

For example, a variant with an amino acid change to proline at aposition in a human IgG4 Fc sequence, in which the C-terminal lysine isabsent (IgG4 ΔK Fc), is shown bolded in the amino acid sequence providedbelow:

(SEQ ID NO: 2005) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

For example, a variant with an amino acid change to Glutamine at aposition in the human IgG1 Fc sequence is shown bolded in the amino acidsequence provided below:

(SEQ ID NO: 2006) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Any of the VH domains of Tables 1-24 and of FIGS. 4A, 5A, 5C, 5E, 6A,7A, 8A, 9A, and 10A may be combined with a variant Fc region describedherein. Exemplary heavy chain constructs comprising a variant Fc regionmay include the following constructs designated as shown below; thevariable region sequence is bolded with CDR sequences underlined:

3P10 Fab Hc: (SEQ ID NO: 1824) QIQLVQSGPELKKPGETVKISCKAS GYTFTDYGVIWVKQAPGKALKWM G WINTYTGEPTYADDLKG RFAFSLETSASSASLQINNLKNEDTATYFCA RRYGPEDIDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVD 25M22 Fab Hc: (SEQ ID NO: 1826)QVQLQQSGPDLVKPGASVKISCKAS GYTFTSYWVN WMKQRPGKGLE WIG RIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVY FCAR AYLLRLRRTGYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVD 8D8 Fab Hc: (SEQ ID NO: 1828)QVQLKESGPGLVAPSQSLSITCTVS GFSLSRYSVH WVRQPPGKGLEW LG MIWGFGSTDYNSALKSRLSITKDNSKSQFFLKMNSLQTDDTAMYYC AR IHTTAGSYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVDG 5F12 Fab Hc: (SEQ ID NO: 1830)QVQLKQSGTELVRPGASVKLSCKAS GYTFTDYYIN WVKQRPGQGLEW IA RIYPGNGNTYHNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFC AR EGLYYDYDRYFDYWGQGTALTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVDG

A “light chain constant region” includes kappa and lambda constantregions. Any of the VL domains of Tables Tables 1-24 and of FIGS. 4B,5B, 5D, 5F, 6B, 7B, 8B, 9B, and 10B may be combined with a kappa orlambda constant region described herein.

An exemplary kappa constant region is provided below:

(SEQ ID NO: 2007) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

An exemplary lambda constant region is provided below:

(SEQ ID NO: 2008) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS

Exemplary light chain constructs comprising a constant region mayinclude the following constructs designated as shown below; the variableregion sequence is bolded with CDR sequences underlined:

3P10 Fab Lc: (SEQ ID NO: 1825) DIVLTQSPVSLAVSLGQRATISC RASESVDNYGISFMSWFQQKPGQPP KLLIY AASHQGS GVPARFSGSGSGTDFSLNIHPMEEDDSAMYFC LQSK EVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25M22 Fab Lc: (SEQ ID NO: 1827)DVVLTQTPLSLPVNIGDQASISC KSTKSLLNSDEFTYLD WYLQKPGQSP QLLIF LVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQSNY LPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC8D8 Fab Lc: (SEQ ID NO: 1829) DIVMTQSQKFMSTSIGDRVSVTC KASQNVGTNVAWYQQKPGQSPKAL VY STSYRYS GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC HQYNSYPL TFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC5F12 Fab Lc: (SEQ ID NO: 1831)NIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQPP KLLIY LASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYC HQNNE DPPAFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

The term “variant” when used in relation to GFRAL or to an anti-GFRALantibody may refer to a peptide or polypeptide comprising one or more(such as, for example, about 1 to about 25, about 1 to about 20, about 1to about 15, about 1 to about 10, or about 1 to about 5) amino acidsequence substitutions, deletions, and/or additions as compared to anative or unmodified GFRAL sequence. For example, a GFRAL variant mayresult from one or more (such as, for example, about 1 to about 25,about 1 to about 20, about 1 to about 15, about 1 to about 10, or about1 to about 5) changes to an amino acid sequence of a native GFRAL. Alsoby way of example, a variant of an anti-GFRAL antibody may result fromone or more (such as, for example, about 1 to about 25, about 1 to about20, about 1 to about 15, about 1 to about 10, or about 1 to about 5)changes to an amino acid sequence of a native or previously unmodifiedanti-GFRAL antibody. Variants may be naturally occurring, such asallelic or splice variants, or may be artificially constructed.Polypeptide variants may be prepared from the corresponding nucleic acidmolecules encoding the variants. In some embodiments, the GFRAL variantor anti-GFRAL antibody variant at least retains GFRAL or anti-GFRALantibody functional activity, respectively. In some embodiments, ananti-GFRAL antibody variant binds GFRAL and/or is antagonistic to GFRALactivity. In some embodiments, an anti-GFRAL antibody variant bindsGFRAL and/or is agonistic to GFRAL activity. In some embodiments, thevariant is encoded by a single nucleotide polymorphism (SNP) variant ofa nucleic acid molecule that encodes GFRAL or anti- GFRAL antibody VH orVL regions or subregions, such as one or more CDRs.

The term “vector” refers to a substance that is used to carry or includea nucleic acid sequences, including for example, in order to introduce anucleic acid sequence into a host cell. Vectors applicable for useinclude, for example, expression vectors, plasmids, phage vectors, viralvectors, episomes and artificial chromosomes, which can includeselection sequences or markers operable for stable integration into ahost cell's chromosome. Additionally, the vectors can include one ormore selectable marker genes and appropriate expression controlsequences. Selectable marker genes that can be included, for example,provide resistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more nucleic acidmolecules are to be co-expressed (e.g. both an antibody heavy and lightchain or an antibody VH and VL) both nucleic acid molecules can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The introductionof nucleic acid molecules into a host cell can be confirmed usingmethods well known in the art. Such methods include, for example,nucleic acid analysis such as Northern blots or polymerase chainreaction (PCR) amplification of mRNA, or immunoblotting for expressionof gene products, or other suitable analytical methods to test theexpression of an introduced nucleic acid sequence or its correspondinggene product. It is understood by those skilled in the art that thenucleic acid molecules are expressed in a sufficient amount to produce adesired product (e.g. an anti-GFRAL antibody as described herein), andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis known (see, e.g., Table 3, page 464, Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991)). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, (see, e.g., U.S. Pat. Nos. 5,500,362or 5,821,337) may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, for example, in a animalmodel (see, e.g., Clynes et al. (USA) 95:652-656 (1998)). Antibodieswith little or no ADCC activity may be selected for use.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one that binds an IgG antibody (e.g., agamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof (see, e.g., review Daëron, Annu. Rev. Immunol. 15:203-234(1997)). FcRs are known (see, e.g., Ravetch and Kinet, Annu. Rev.Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994);and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995)). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(see, e.g., Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)). Antibody variants with improved or diminishedbinding to FcRs have been described (see, e.g., in WO 2000/42072; U.S.Pat. Nos. 7,183,387, 7,332,581 and 7.335,742; Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001)).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, (see, e.g., Gazzano-Santoro et al., J. Immunol.Methods 202:163 (1996)), may be performed. Polypeptide variants withaltered Fc region amino acid sequences (polypeptides with a variant Fcregion) and increased or decreased Cl q binding capability have beendescribed, (see, e.g., U.S. Pat. No. 6,194,551, WO 1999/51642, Idusogieet al. J. Immunol. 164: 4178-4184 (2000)). Antibodies with little or noCDC activity may be selected for use.

In calculating percent identity, the sequences being compared may bealigned in a way that gives the largest match between the sequences.Computer program may be used to determine percent identity is the GCGprogram package, which includes GAP (Devereux et al., (1984) Nucl. AcidRes. 12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP used to align the two polypeptides orpolynucleotides for which the percent sequence identity is to bedetermined. The sequences may be aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as3.times. the average diagonal, wherein the “average diagonal” is theaverage of the diagonal of the comparison matrix being used; the“diagonal” is the score or number assigned to each perfect amino acidmatch by the particular comparison matrix) and a gap extension penalty(which is usually 1/10 times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. In certain embodiments, a standard comparison matrix(see, Dayhoff et al., (1978) Atlas of Protein Sequence and Structure5:345-352 for the PAM 250 comparison matrix; Henikoff et al., (1992)Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62comparison matrix) is also used by the algorithm.

Examplary parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following: (i)Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; (ii)Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra; (iii)Gap Penalty: 12 (but with no penalty for end gaps) (iv) Gap LengthPenalty: 4; and (v) Threshold of Similarity: 0.

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if so desired to result in an alignment thatspans a number of amino acids, for example, at least 50 contiguous aminoacids of the target polypeptide.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

A “modification” of an amino acid residue/position refers to a change ofa primary amino acid sequence as compared to a starting amino acidsequence, wherein the change results from a sequence alterationinvolving said amino acid residue/positions. For example, typicalmodifications include substitution of the residue with another aminoacid (e.g., a conservative or non-conservative substitution), insertionof one or more (e.g., generally fewer than 5, 4 or 3) amino acidsadjacent to said residue/position, and/or deletion of saidresidue/position.

An “epitope” is the site on the surface of an antigen molecule to whicha single antibody molecule binds, such as a localized region on thesurface of an antigen, such as a GFRAL polypeptide, a GFRAL polypeptidefragment or a GFRAL epitope, that is capable of being bound to one ormore antigen binding regions of an antibody, and that has antigenic orimmunogenic activity in an animal, such as a mammal (e.g., a human),that is capable of eliciting an immune response. An epitope havingimmunogenic activity is a portion of a polypeptide that elicits anantibody response in an animal. An epitope having antigenic activity isa portion of a polypeptide to which an antibody binds as determined byany method well known in the art, including, for example, by animmunoassay. Antigenic epitopes need not necessarily be immunogenic.Epitopes often consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics. The term, “epitope” specifically includes linearepitopes and conformational epitopes. A region of a polypeptidecontributing to an epitope may be contiguous amino acids of thepolypeptide or the epitope may come together from two or morenon-contiguous regions of the polypeptide. The epitope may or may not bea three-dimensional surface feature of the antigen. In certainembodiments, a GFRAL epitope is a three-dimensional surface feature of aGFRAL polypeptide. In other embodiments, a GFRAL epitope is linearfeature of a GFRAL polypeptide. Generally an antigen has several or manydifferent epitopes and may react with many different antibodies.

An antibody binds “an epitope” or “essentially the same epitope” or “thesame epitope” as a reference antibody, when the two antibodies recognizeidentical, overlapping or adjacent epitopes in a three-dimensionalspace. The most widely used and rapid methods for determining whethertwo antibodies bind to identical, overlapping or adjacent epitopes in athree-dimensional space are competition assays, which can be configuredin a number of different formats, for example, using either labeledantigen or labeled antibody. In some assays, the antigen is immobilizedon a 96-well plate, or expressed on a cell surface, and the ability ofunlabeled antibodies to block the binding of labeled antibodies ismeasured using radioactive, fluorescent or enzyme labels.

“Epitope mapping” is the process of identifying the binding sites, orepitopes, of antibodies on their target antigens. Antibody epitopes maybe linear epitopes or conformational epitopes. Linear epitopes areformed by a continuous sequence of amino acids in a protein.Conformational epitopes are formed of amino acids that are discontinuousin the protein sequence, but which are brought together upon folding ofthe protein into its three-dimensional structure. Induced epitopes areformed when the three dimensional structure of the protein is in analtered confirmation, such as following activation or binding of anotherprotein or ligand.

“Epitope binning” is the process of grouping antibodies based on theepitopes they recognize. More particularly, epitope binning comprisesmethods and systems for discriminating the epitope recognitionproperties of different antibodies, using competition assays combinedwith computational processes for clustering antibodies based on theirepitope recognition properties and identifying antibodies havingdistinct binding specificities.

A “GFRAL-mediated disease,” “GFRAL-mediated disorder,” and“GFRAL-mediated condition” are used interchangeably and refer to anydisease, disorder or condition that is completely or partially caused byor is the result of GFRAL or the interaction of a GFRAL with GDF15and/or alternatively any disease, disorder, or condition in which it isdesirable to inhibit the in vivo effects of GDF15. GFRAL-mediateddiseases, disorders, or conditions include GDF15-mediated diseasesdisorders or conditions.

A “GDF15-mediated disease,” “GDF15-mediated disorder,” and“GDF15-mediated condition” are used interchangeably and refer to anydisease, disorder or condition that is: (i) completely or partiallycaused by; or (ii) is the result of a GDF15 protein (e.g., an activityof a GDF15 protein, such as GDF15 signaling or elevated levels of aGDF15 protein) or the interaction of a GFRAL protein with a GDF15protein and/or a RET protein, alternatively any disease, disorder, orcondition in which it is desirable to inhibit the in vivo effects ofGDF15. GDF15-mediated diseases, disorders, or conditions includeinvoluntary weight loss, cachexia, sarcopenia, muscle wasting, bonewasting, a cardiovascular disease, a chronic inflammatory disease (e.g.,chronic renal disease, chronic obstructive pulmonary disease), and acancer, including a cancer that has decreased sensitivity to (e.g.,resistance to) a chemotherapeutic agent (e.g., an anti-tumor antibodysuch as trastuzumab) that is induced by or related to a GDF15 protein.

The term “therapeutically effective amount” as used herein refers to theamount of an agent (e.g., an antibody described herein or any otheragent described herein) that is sufficient to reduce and/or amelioratethe severity and/or duration of a given disease, disorder or condition,and/or a symptom related thereto. A therapeutically effective amount ofa agent, including a therapeutic agent, can be an amount necessary for(i) reduction or amelioration of the advancement or progression of agiven disease, disorder, or condition, (ii) reduction or amelioration ofthe recurrence, development or onset of a given disease, disorder orconditions, and/or (iii) to improve or enhance the prophylactic ortherapeutic effect of another therapy (e.g., a therapy other than theadministration of an antibody provided herein). A “therapeuticallyeffective amount” of a substance/molecule/agent of the presentdisclosure (e.g., an anti-GFRAL antibody) may vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe ability of the substance/molecule/agent, to elicit a desiredresponse in the individual. A therapeutically effective amountencompasses an amount in which any toxic or detrimental effects of thesubstance/molecule/agent are outweighed by the therapeuticallybeneficial effects. In certain embodiments, the term “therapeuticallyeffective amount” refers to an amount of an antibody or other agent(e.g., or drug) effective to “treat” a disease, disorder, or condition,in a subject or mammal.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofa disease, disorder, or condition, a prophylactically effective amountmay be less than a therapeutically effective amount.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode (e.g., for a period of time such as days, weeks, monthsor years) as opposed to an acute mode, so as to maintain the initialtherapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further agents includessimultaneous (e.g., concurrent) and consecutive administration in anyorder. The term “in combination” in the context of the administration ofother therapies (e.g., other agents) includes the use of more than onetherapy (e.g., one agent). The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject. Afirst therapy (e.g., agent) can be administered before (e.g., 1 minute,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently,or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours,24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,or 12 weeks) the administration of a second therapy (e.g., agent) to asubject which had, has, or is susceptible to or has a risk of aGDF15-mediated disease, disorder or condition.

Any additional therapy (e.g., agent) can be administered in any orderwith the other additional therapies (e.g., agents). In certainembodiments, the antibodies can be administered in combination with oneor more therapies such as agents (e.g., therapies, including agents,that are not the antibodies that are currently administered) to prevent,treat, manage, and/or ameliorate a GDF15-mediated disease, disorder orcondition, or a symptom thereof. Non-limiting examples of therapies(e.g., agents) that can be administered in combination with an antibodyinclude, for example, analgesic agents, anesthetic agents, antibiotics,or immunomodulatory agents or any other agent listed in the U.S.Pharmacopoeia and/or Physician's Desk Reference. Examples of agentsuseful in combination therapy include, but are not limited to, thefollowing: non-steroidal anti-inflammatory drug (NSAID) such as aspirin,ibuprofen, and other propionic acid derivatives (alminoprofen,benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen,flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac,ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin,and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone).Other combinations include cyclooxygenase-2 (COX-2) inhibitors. Otheragents for combination include steroids such as prednisolone,prednisone, methylprednisolone, betamethasone, dexamethasone, orhydrocortisone. Such a combination may be especially advantageous, sinceone or more side-effects of the steroid can be reduced or eveneliminated by tapering the steroid dose required when treating subjectsin combination with the present antibodies. Additional examples ofagents for combinations include cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, TNF, LT, IL-1β, IL-2, IL-6, IL-7, IL-8,IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, or PDGF. Combinations ofagents may include TNF antagonists like chimeric, humanized or human TNFantibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870), andsoluble p55 or p75 TNF receptors, derivatives thereof, p75TNFRIgG(ENBREL®) or p55TNFR1gG (LENERCEPT®), soluble IL-13 receptor (sIL-13),and also TNFα converting enzyme (TACE) inhibitors; similarly IL-1inhibitors (e.g., Interleukin-1-converting enzyme inhibitors) may beeffective. Other combinations include Interleukin 11, anti-P7s andp-selectin glycoprotein ligand (PSGL). Other examples of agents usefulin combination therapy include interferon-β1a (AVONEX); interferon-β1b(BETASERON®); copaxone; hyperbaric oxygen; intravenous immunoglobulin;clabribine; and antibodies to or antagonists of other human cytokines orgrowth factors (e.g., antibodies to CD40 ligand and CD80).

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight ((e.g., less than about 10 aminoacid residues) polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™. The term “carrier” can also refer to a diluent,adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient,or vehicle with which the therapeutic is administered. Such carriers,including pharmaceutical carriers, can be sterile liquids, such as waterand oils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water is a exemplary carrier when a composition (e.g., apharmaceutical composition) is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable excipients (e.g., pharmaceutical excipients) include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like. Oralcompositions, including formulations, can include standard carriers suchas pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, etc.Examples of suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton,Pa. Compositions, including pharmaceutical compounds, may contain aprophylactically or therapeutically effective amount of an anti-GFRALantibody, for example, in isolated or purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the subject (e.g., patient). The formulation shouldsuit the mode of administration.

The term “pharmaceutically acceptable” as used herein means beingapproved by a regulatory agency of the Federal or a state government, orlisted in the U.S. Pharmacopeia, European Pharmacopeia or othergenerally recognized Pharmacopeia for use in animals, and moreparticularly in humans.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient (e.g., an anti- GFRAL antibody) to be effective, and whichcontains no additional components which are unacceptably toxic to asubject to which the formulation would be administered. Such formulationmay be sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

“Polyclonal antibodies” as used herein refers to an antibody populationgenerated in an immunogenic response to a protein having many epitopesand thus includes a variety of different antibodies directed to the sameand to different epitopes within the protein. Methods for producingpolyclonal antibodies are known in the art (See, e.g., Chapter 11 in:Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al.,eds., John Wiley and Sons, N.Y.).

An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA,or a mixed polymer, which is substantially separated from other genomeDNA sequences as well as proteins or complexes such as ribosomes andpolymerases, which naturally accompany a native sequence. An “isolated”nucleic acid molecule is one which is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acidmolecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. In a specific embodiment, one or more nucleic acidmolecules encoding an antibody as described herein are isolated orpurified. The term embraces nucleic acid sequences that have beenremoved from their naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analoguesor analogues biologically synthesized by heterologous systems. Asubstantially pure molecule may include isolated forms of the molecule.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. “Oligonucleotide,” as usedherein, generally refers to short, generally single-stranded, generallysynthetic polynucleotides that are generally, but not necessarily, lessthan about 200 nucleotides in length. The terms “oligonucleotide” and“polynucleotide” are not mutually exclusive. The description above forpolynucleotides is equally and fully applicable to oligonucleotides. Acell that produces an anti-GFRAL antibody of the present disclosure mayinclude a parent hybridoma cell, as well as bacterial and eukaryotichost cells into which nucleic acid encoding the antibodies have beenintroduced. Suitable host cells are disclosed below.

As used herein, the terms “treat,” “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity, and/orduration of a GDF15-mediated disease, disorder or condition resultingfrom the administration of one or more therapies (including, but notlimited to, the administration of one or more prophylactic ortherapeutic agents, such as an antibody provided herein). In someembodiments, the agent is an anti-GFRAL antibody. Treatment as usedherein includes, but is not limited to, (i) increase body weight; (ii)maintain body weight; (iii) reduce body weight loss; (iv) increase bodymass (e.g., lean mass or fat mass); (v) maintain body mass (e.g., leanmass or fat mass); (vi) reduce loss of body mass (e.g., lean mass or fatmass), or any combination thereof.

As used herein, the term “prophylactic agent” refers to any agent thatcan totally or partially inhibit the development, recurrence, onset orspread of a GDF15-mediated disease, disorder or condition and/or symptomrelated thereto in a subject. In some embodiments, the term“prophylactic agent” refers to an antibody provided herein. In someembodiments, the term “prophylactic agent” refers to an agent other thanan antibody provided herein. In some embodiments, a prophylactic agentis an agent which is known to be useful to or has been or is currentlybeing used to prevent a GDF15-mediated disease, disorder or conditionand/or a symptom related thereto or impede the onset, development,progression and/or severity of a GDF15-mediated disease, disorder orcondition and/or a symptom related thereto. In some embodiments, theprophylactic agent is a fully human anti-GFRAL antibody, such as a fullyhuman anti-GFRAL monoclonal antibody.

The term “prophylactic agent” refers to any agent that can totally orpartially inhibit the development, recurrence, onset or spread of aGDF15-mediated disease, disorder or condition, or a symptom thereof in asubject. In certain embodiments, the term “prophylactic agent” refers toan anti-GFRAL antibody as described herein. In certain otherembodiments, the term “prophylactic agent” refers to an agent other thanan anti-GFRAL antibody as described herein. In certain embodiments, aprophylactic agent is an agent which is known to be useful to or hasbeen or is currently being used to prevent a GDF15-mediated disease,disorder or condition, or a symptom thereof or impede the onset,development, progression and/or severity of a GDF15-mediated disease,disorder or condition, or a symptom thereof. In specific embodiments,the prophylactic agent is a humanized anti-GFRAL antibody, such as ahumanized anti-GFRAL monoclonal antibody.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The terms “prevent,” “preventing,” and “prevention” refer to the totalor partial inhibition of the development, recurrence, onset or spread ofa GDF15-mediated disease, disorder or condition, or a symptom thereof,resulting from the administration of a therapy or combination oftherapies provided herein (e.g., a combination of prophylactic ortherapeutic agents, such as an antibody provided herein).

The term “recombinant antibody” refers to an antibody that is prepared,expressed, created or isolated by recombinant means. Recombinantantibodies can be antibodies expressed using a recombinant expressionvector transfected into a host cell, antibodies isolated from arecombinant, combinatorial antibody library, antibodies isolated from ananimal (e.g., a mouse or cow) that is transgenic and/or transchromosomalfor human immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992)Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed,created or isolated by any other means that involves splicing ofimmunoglobulin gene sequences to other DNA sequences. Such recombinantantibodies can have variable and constant regions, including thosederived from human germline immunoglobulin sequences (See Kabat, E. A.et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242). In certain embodiments, however, such recombinantantibodies may be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germ line VH and VL sequences, may not naturally existwithin the human antibody germ line repertoire in vivo.

The term “side effects” encompasses unwanted and adverse effects of atherapy (e.g., a prophylactic or therapeutic agent). Unwanted effectsare not necessarily adverse.An adverse effect from a therapy (e.g., aprophylactic or therapeutic agent) might be harmful or uncomfortable orrisky. Examples of side effects include, diarrhea, cough,gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects,fatigue, dry mouth, and loss of appetite, rashes or swellings at thesite of administration, flu-like symptoms such as fever, chills andfatigue, digestive tract problems and allergic reactions. Additionalundesired effects experienced by patients are numerous and known in theart. Many are described in the Physician's Desk Reference (60th ed.,2006).

The terms “subject” and “patient” are used interchangeably herein and,in the context of the methods disclosed herein, refer to an animal thatis the recipient of a therapy or preventive case. As used herein, incertain embodiments, a subject is a mammal, such as a non-primate (e.g.,cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeyand human). In specific embodiments, the subject is a human. In oneembodiment, the subject is a mammal (e.g., a human) having aGDF15-mediated disease, disorder or condition. In another embodiment,the subject is a mammal (e.g., a human) at risk of developing aGDF15-mediated disease, disorder or condition.

“Substantially all” refers to refers to at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100%.

The term “therapeutic agent” refers to any agent that can be used intreating, preventing or alleviating a disease, disorder or condition,including in the treatment, prevention or alleviation of one or moresymptoms of a GDF15-mediated disease, disorder or condition, or asymptom thereof. In certain embodiments, a therapeutic agent refers toan anti-GFRAL antibody as described herein. In certain otherembodiments, a therapeutic agent refers to an agent other than anantibody provided herein. In certain embodiments, a therapeutic agent isan agent which is known to be useful for, or has been or is currentlybeing used for the treatment, prevention or alleviation of one or moresymptoms of a GDF15-mediated disease, disorder or condition, or asymptom thereof.

The combination of therapies (e.g., use of prophylactic or therapeuticagents) which is more effective than the additive effects of any two ormore single therapy. For example, a synergistic effect of a combinationof prophylactic and/or therapeutic agents permits the use of lowerdosages of one or more of the agents and/or less frequent administrationof the agents to a subject with a GDF15-mediated disease, disorder orcondition. The ability to utilize lower dosages of prophylactic ortherapeutic therapies and/or to administer the therapies less frequentlyreduces the toxicity associated with the administration of the therapiesto a subject without reducing the efficacy of the therapies in theprevention, management, treatment or amelioration of a GDF15-mediateddisease, disorder or condition. In addition, a synergistic effect canresult in improved efficacy of therapies in the prevention, or in themanagement, treatment or amelioration of a GDF15-mediated disease,disorder or condition. Finally, synergistic effect of a combination oftherapies (e.g., prophylactic or therapeutic agents) can avoid or reduceadverse or unwanted side effects associated with the use of any singletherapy. In some embodiments, the combination therapy comprises anantibody provided herein and insulin (e.g., insulin supplementation). Insome embodiments, the combination therapy comprises an-anti-GFRALantibody and insulin, wherein the combination therapy comprises insulinat a lower daily dosage than the normal daily dosage in an insulin-onlytherapy. In some embodiments, the combination therapy comprises, e.g.,less than 90%, less than 80%, less than 70%, less than 60%, less than50%, less than 40%, less than 30%, less than 20%, less than 15%, lessthan 10%, less than 5%, less than 3%, or less than 1% of the normaldaily insulin dosage in an insulin-only therapy.

The term “therapy” refers to any protocol, method and/or agent that canbe used in the prevention, management, treatment and/or amelioration ofa GDF15-mediated disease, disorder or condition (e.g., type 1 diabetesor type 2 diabetes). In some embodiments, the terms “therapies” and“therapy” refer to a biological therapy, supportive therapy, and/orother therapies useful in the prevention, management, treatment and/oramelioration of a GDF15-mediated disease, disorder or condition known toone of skill in the art such as medical personnel.

The term “detectable probe” refers to a composition that provides adetectable signal. The term includes, without limitation, anyfluorophore, chromophore, radiolabel, enzyme, antibody or antibodyfragment, and the like, that provide a detectable signal via itsactivity.

The term “diagnostic agent” refers to a substance administered to asubject that aids in the diagnosis of a disease, disorder, orconditions. Such substances can be used to reveal, pinpoint, and/ordefine the localization of a disease causing process. In certainembodiments, a diagnostic agent includes a substance that is conjugatedto an anti-GFRAL antibody as described herein, that when administered toa subject or contacted to a sample from a subject aids in the diagnosisa GDF15-mediated disease, disorder or condition.

The term “detectable agent” refers to a substance that can be used toascertain the existence or presence of a desired molecule, such as ananti-GFRAL antibody as described herein, in a sample or subject. Adetectable agent can be a substance that is capable of being visualizedor a substance that is otherwise able to be determined and/or measured(e.g., by quantitation).

The term “encode” or grammatical equivalents thereof as it is used inreference to nucleic acid molecule refers to a nucleic acid molecule inits native state or when manipulated by methods well known to thoseskilled in the art that can be transcribed to produce mRNA, which isthen translated into a polypeptide and/or a fragment thereof. Theantisense strand is the complement of such a nucleic acid molecule, andthe encoding sequence can be deduced therefrom.

The term “excipient” refers to an inert substance which is commonly usedas a diluent, vehicle, preservative, binder, or stabilizing agent, andincludes, but not limited to, proteins (e.g., serum albumin, etc.),amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine,glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkylsulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate,nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose,trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See,also, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co.,Easton, Pa., which is hereby incorporated by reference in its entirety.

In the context of a peptide or polypeptide, the term “fragment” as usedherein refers to a peptide or polypeptide that comprises less than thefull length amino acid sequence. Such a fragment may arise, for example,from a truncation at the amino terminus, a truncation at the carboxyterminus, and/or an internal deletion of a residue(s) from the aminoacid sequence. Fragments may, for example, result from alternative RNAsplicing or from in vivo protease activity. In certain embodiments,fragments include polypeptides comprising an amino acid sequence of atleast 5 contiguous amino acid residues, at least 10 contiguous aminoacid residues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at leastcontiguous 100 amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, at least 250, at least 300, at least 350, at least 400, atleast 450, at least 500, at least 550, at least 600, at least 650, atleast 700, at least 750, at least 800, at least 850, at least 900, or atleast 950, contiguous amino acid residues of the amino acid sequence ofa GFRAL polypeptide or an antibody that binds to a GFRAL polypeptide. Insome embodiments, a fragment of an antibody that binds to a GFRALpolypeptide retains at least 1, at least 2, or at least 3 or morefunctions of the antibody.

The terms “manage,” “managing,” and “management” refer to the beneficialeffects that a subject derives from a therapy (e.g., a prophylactic ortherapeutic agent), which does not result in a cure of the disease. Incertain embodiments, a subject is administered one or more therapies(e.g., prophylactic or therapeutic agents, such as an antibody providedherein) to “manage” a GDF15-mediated disease, disorder or condition, ora symptom thereof, so as to prevent the progression or worsening of thedisease.

“Administer” or “administration” refers to the act of injecting orotherwise physically delivering a substance as it exists outside thebody (e.g., an anti-GFRAL antibody as described herein) into a subject,such as by mucosal, intradermal, intravenous, intramuscular deliveryand/or any other method of physical delivery described herein or knownin the art. When a disease, disorder, or condition, or a symptomthereof, is being treated, administration of the substance typicallyoccurs after the onset of the disease, disorder, or condition, orsymptoms thereof. When a disease, disorder, or condition or symptomsthereof, are being prevented, administration of the substance typicallyoccurs before the onset of the disease, disorder, or condition, orsymptoms thereof.

In the context of a polypeptide, the term “analog” as used herein refersto a polypeptide that possesses a similar or identical function as aGFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRALantibody but does not necessarily comprise a similar or identical aminoacid sequence of a GFRAL polypeptide, a fragment of a GFRAL polypeptide,or an anti-GFRAL antibody, or possess a similar or identical structureof a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or ananti-GFRAL antibody. A polypeptide that has a similar amino acidsequence refers to a polypeptide that satisfies at least one of thefollowing: (a) a polypeptide having an amino acid sequence that is atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of a GFRAL polypeptide (e.g., SEQID NO:500, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibodydescribed herein; (b) a polypeptide encoded by a nucleotide sequencethat hybridizes under stringent conditions to a nucleotide sequenceencoding a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or ananti-GFRAL antibody (or VH or VL region thereof) described herein of atleast 5 amino acid residues, at least 10 amino acid residues, at least15 amino acid residues, at least 20 amino acid residues, at least 25amino acid residues, at least 40 amino acid residues, at least 50 aminoacid residues, at least 60 amino residues, at least 70 amino acidresidues, at least 80 amino acid residues, at least 90 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, or at least 150 amino acid residues (see, e.g., Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Maniatis et al. (1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.); and (c) a polypeptide encoded by a nucleotidesequence that is at least 30%, at least 35%, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the nucleotide sequence encoding a GFRALpolypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRALantibody (or VH or VL region thereof) described herein. A polypeptidewith similar structure to a GFRAL polypeptide, a fragment of a GFRALpolypeptide, or an anti-GFRAL antibody described herein refers to apolypeptide that has a similar secondary, tertiary or quaternarystructure of a GFRAL polypeptide, a fragment of a GFRAL, or a GFRALantibody described herein. The structure of a polypeptide can determinedby methods known to those skilled in the art, including but not limitedto, X-ray crystallography, nuclear magnetic resonance, andcrystallographic electron microscopy.

The term “composition” is intended to encompass a product containing thespecified ingredients (e.g., an antibody provided herein) in,optionally, the specified amounts, as well as any product which results,directly or indirectly, from combination of the specified ingredientsin, optionally, the specified amounts.

In the context of a polypeptide, the term “derivative” as used hereinrefers to a polypeptide that comprises an amino acid sequence of a GFRALpolypeptide, a fragment of a GFRAL polypeptide, or an antibody thatbinds to a GFRAL polypeptide which has been altered by the introductionof amino acid residue substitutions, deletions or additions. The term“derivative” as used herein also refers to a GFRAL polypeptide, afragment of a GFRAL polypeptide, or an antibody that binds to a GFRALpolypeptide which has been chemically modified, e.g., by the covalentattachment of any type of molecule to the polypeptide. For example, butnot by way of limitation, a GFRAL polypeptide, a fragment of a GFRALpolypeptide, or a GFRAL antibody may be chemically modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Thederivatives are modified in a manner that is different from naturallyoccurring or starting peptide or polypeptides, either in the type orlocation of the molecules attached. Derivatives further include deletionof one or more chemical groups which are naturally present on thepeptide or polypeptide. A derivative of a GFRAL polypeptide, a fragmentof a GFRAL polypeptide, or a GFRAL antibody may be chemically modifiedby chemical modifications using techniques known to those of skill inthe art, including, but not limited to specific chemical cleavage,acetylation, formulation, metabolic synthesis of tunicamycin, etc.Further, a derivative of a GFRAL polypeptide, a fragment of a GFRALpolypeptide, or a GFRAL antibody may contain one or more non-classicalamino acids. A polypeptide derivative possesses a similar or identicalfunction as a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or aGFRAL antibody described herein.

The term “involuntary body weight loss” refers to the unintended loss ofbody weight that is observed in many conditions such as cachexia, livercirrhosis, hyperthyroidism, chronic kidney disease, Parkinson's disease,cancer, eating disorder, and sarcopenia.

The term “cachexia” refers to wasting syndrome that is marked with lossof weight, muscle atrophy, fatigue, weakness, and significant loss ofappetite in someone who is not actively trying to lose weight. Cachexiacan greatly contribute to morbidity of patients suffering from somechronic diseases (e.g., cancer, chronic renal disease, chronicinflammatory disease, muscle wasting, such as muscular dystrophy, andanorexia nervosa). For example, in late stage cancer, cachexia is common(occurring in most terminally ill cancer patients), and is responsiblefor about a quarter of all cancer-related deaths.

Compositions and Methods of Making the Same

Binding proteins such as antibodies that bind to GFRAL (e.g., humanGFRAL) are provided. Antibodies of the present disclosure are useful,for example, for the diagnosis or treatment of GDF15-mediated diseases,disorders, or conditions. In certain embodiments, antibodies of thepresent disclosure are useful for the diagnosis or treatment of adisease, disorder, or condition, such as involuntary body weight loss,including, but not limited to, involuntary body weight loss in a subjectsuffering from cachexia or a chronic disease (e.g., liver cirrhosis,hyperthyroidism, Parkinson's disease, cancer, chronic renal disease,chronic obstructive pulmonary disease, AIDS, tuberculosis, chronicinflammatory disease, sepsis, muscle wasting, and anorexia nervosa) orbroadly any disease, disorder, or condition in which it is desirable toinhibit the in vivo effects of GDF15.

Provided herein are antibodies (e.g., monoclonal antibodies) that bindto a GFRAL polypeptide, a GFRAL polypeptide fragment, GFRAL peptide, ora GFRAL epitope. In some embodiments, the anti-GFRAL antibodies bind tothe extracellular domain (ECD) of GFRAL. Also provided are antibodiesthat competitively block an anti-GFRAL antibody provided herein frombinding to a GFRAL polypeptide. The anti-GFRAL antibodies providedherein can also be conjugated or recombinantly fused to a diagnosticagent, detectable agent or therapeutic agent. Further provided arecompositions comprising an anti-GFRAL antibody.

Also provided herein are isolated nucleic acid molecules encoding animmunoglobulin heavy chain, light chain, VH region, VL region, VH CDR1,VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of anti-GFRALantibodies that bind to a GFRAL polypeptide, a GFRAL polypeptidefragment, a GFRAL peptide or a GFRAL epitope. Further provided arevectors and host cells comprising nucleic acid molecules encodinganti-GFRAL antibodies that bind to a GFRAL polypeptide, a GFRALpolypeptide fragment, a GFRAL peptide or a GFRAL epitope. Also providedare methods of making antibodies that bind to a GFRAL polypeptide, aGFRAL polypeptide fragment, a GFRAL peptide or a GFRAL epitope.

Methods of using the anti-GFRAL antibodies are provided herein. Themethods include treating, preventing or alleviating a GDF15-mediateddisease, disorder or condition, including treating, preventing oralleviating one or more symptoms of a GDF15-mediated disease, disorderor condition in a subject (e.g., patient). Non limiting examples ofGDF15-mediated diseases, disorders, or conditions include involuntaryweight loss, a waisting disease, involuntary body weight loss in asubject suffering from cachexia or a chronic disease (e.g., livercirrhosis, hyperthyroidism, Parkinson's disease, cancer, chronic renaldisease, chronic obstructive pulmonary disease, AIDS, tuberculosis,chronic inflammatory disease, sepsis, muscle wasting, and anorexianervosa). Other of diseases, disorders, or conditions in which a subjectcan suffer from involuntary weight loss include eating disorders,muscular dystrophy or multiple sclerosis.

Anti-GFRAL Antibodies

In some embodiments, the present disclosure provides anti-GFRALantibodies that may find use herein as therapeutic agents. Exemplaryantibodies include polyclonal, monoclonal, humanized, human, bispecific,and heteroconjugate antibodies, as well as variants thereof havingimproved affinity or other properties.

In some embodiments, provided herein are antibodies that bind to GFRAL,including a GFRAL polypeptide, a GFRAL polypeptide fragment, a GFRALpeptide or a GFRAL epitope. In some embodiments the anti-GFRALantibodies are humanized antibodies (e.g., comprising human constantregions) that bind GFRAL, including GFRAL polypeptide, a GFRALpolypeptide fragment, a GFRAL peptide or a GFRAL epitope.

In some embodiments, an anti-GFRAL antibody comprises a VH region, VLregion, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 ofany one of the monoclonal antibodies described herein (e.g., 1C1, 3P10,12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19,2B3, 8C10, 2A9, 24G2, 6G9, 2B11,1A3, P1B6, P1H8, or P8G4), such as anamino acid sequence depicted in Tables 1-24. Accordingly, in someembodiments, the isolated antibody or functional fragment thereofprovided herein comprises one, two, and/or three heavy chain CDRs and/orone, two, and/or three light chain CDRs from: (a) the antibodydesignated 1C1; (b) the antibody designated 3P10; (c) the antibodydesignated 12A3; (d) the antibody designated 5F12; (e) the antibodydesignated 5A20; (f) the antibody designated 8D8; (g) the antibodydesignated 17J16; (h) t the antibody designated 25M22; (i) the antibodydesignated 2B8; (j) the antibody designated 22N5; (k) the antibodydesignated 2I23; (l) the antibody designated 6N16; (m) the antibodydesignated 1B3; (n) the antibody designated 19K19; (o) the antibodydesignated 2B3; (p) the antibody designated 8C10; (q) the antibodydesignated 2A9; (r) the antibody designated 24G2; (s) the antibodydesignated 6G9; (t) the antibody designated 2B11; (u) the antibodydesignated 1A3; (v) the antibody designated P1B6; (w) the antibodydesignated P1H8; (x) the antibody designated P8G4; or the antibodydesignated 1C1 to P8G4, as shown in Tables 1-24.

The antibody designated 1C1 comprises a VH sequence that is SEQ ID NO: 1and a VL sequence that is SEQ ID NO: 2.

The antibody designated 3P10 comprises a VH sequence that is SEQ ID NO:3 and a VL sequence that is SEQ ID NO: 4,

The antibody designated 12A3 comprises a VH sequence that is SEQ ID NO:5 and a VL sequence that is SEQ ID NO: 6.

The antibody designated 5F12 comprises a VH sequence that is SEQ ID NO:7 and a VL sequence that is SEQ ID NO: 8.

The antibody designated 5A20 comprises a VH sequence that is SEQ ID NO:9 and a VL sequence that is SEQ ID NO: 10.

The antibody designated 8D8 comprises a VH sequence that is SEQ ID NO:11 and a VL sequence that is SEQ ID NO: 12.

The antibody designated 17J16 comprises a VH sequence that is SEQ ID NO:13 and a VL sequence that is SEQ ID NO: 14.

The antibody designated 25M22 comprises a VH sequence that is SEQ ID NO:15 and a VL sequence that is SEQ ID NO: 16.

The antibody designated 2B8 comprises a VH sequence that is SEQ ID NO:17 and a VL sequence that is SEQ ID NO: 18.

The antibody designated 22N5 comprises a VH sequence that is SEQ ID NO:19 and a VL sequence that is SEQ ID NO: 20.

The antibody designated 2I23 comprises a VH sequence that is SEQ ID NO:21 and a VL sequence that is SEQ ID NO: 22.

The antibody designated 6N16 comprises a VH sequence that is SEQ ID NO:23 and a VL sequence that is SEQ ID NO: 24.

The antibody designated 1B3 comprises a VH sequence that is SEQ ID NO:25 and a VL sequence that is SEQ ID NO: 26.

The antibody designated 19K19 comprises a VH sequence that is SEQ ID NO:27 and a VL sequence that is SEQ ID NO: 28.

The antibody designated 2B3 comprises a VH sequence that is SEQ ID NO:29 and a VL sequence that is SEQ ID NO: 30.

The antibody designated 8C10 comprises a VH sequence that is SEQ ID NO:31 and a VL sequence that is SEQ ID NO: 32.

The antibody designated 2A9 comprises a VH sequence that is SEQ ID NO:33 and a VL sequence that is SEQ ID NO: 34.

The antibody designated 24G2 comprises a VH sequence that is SEQ ID NO:35 and a VL sequence that is SEQ ID NO: 36.

The antibody designated 6G9 comprises a VH sequence that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO: 38.

The antibody designated 2B11 comprises a VH sequence that is SEQ ID NO:39 and a VL sequence that is SEQ ID NO: 40.

The antibody designated 1A3 comprises a VH sequence that is SEQ ID NO:480 and a VL sequence that is SEQ ID NO: 481.

The antibody designated P1B6 comprises a VH sequence that is SEQ ID NO:482 and a VL sequence that is SEQ ID NO: 483.

The antibody designated P1H8 comprises a VH sequence that is SEQ ID NO:484 and a VL sequence that is SEQ ID NO: 485.

The antibody designated P8G4 comprises a VH sequence that is SEQ ID NO:486 and a VL sequence that is SEQ ID NO: 487.

TABLE 1 Antibody 1C1 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH GFSLNDYGVH GFSLNDYG DYGVH GFSLNDY NDYGVH GFSLNDYGVH Seq.CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 43) (SEQ ID NO: 44) (SEQ ID NO: 45)(SEQ ID NO: 41) NO: 41) NO: 42) VH VIWSGGRTDYNA IWSGGRT VIWSGGRTDYNA SGGWLGVIWSGGRTD VIWSGGRTD CDR2 AFIS (SEQ ID AFIS (SEQ ID NO: 134) (SEQ IDNO: 135) (SEQ ID NO: 136) (SEQ ID NO: 133) (SEQ ID NO: 132) NO: 132) VHWALYFLYGGSMDY ARWALYFLYGGS WALYFLYGGSMDY ALYFLYGGSMD ARWALYFLYGGSWALYFLYGGSMDY CDR3 (SEQ ID MDY (SEQ ID NO: 221) (SEQ ID NO: 223) MD (SEQID NO: 221) NO: 221) (SEQ ID (SEQ ID NO: 224) NO: 222) VL CDR VLRSSQSLVHSSGIT QSLVHSSGITY RSSQSLVHSSGIT SQSLVHSSGITY VHSSGITYLHWYRSSQSLVHSSGIT Seq. CDR1 YLH (SEQ ID YLH (SEQ ID NO: 299) (SEQ ID NO:300) YLH (SEQ ID NO: 298) (SEQ ID NO: 297) (SEQ ID NO: 297) NO: 297) VLKLSNRFS KLS KLSNRFS KLS LLIYKLSNRF KLSNRFS CDR2 (SEQ ID (SEQ ID (SEQ IDNO: 373) (SEQ ID NO: 374) (SEQ ID NO: 375) (SEQ ID NO: 373) NO: 373) NO:374) VL SQSTHVPPWT SQSTHVPPWT SQSTHVPPWT STHVPPW SQSTHVPPW SQSTHVPPWTCDR3 (SEQ ID (SEQ ID (SEQ ID NO: 423) (SEQ ID NO: 424) (SEQ ID NO: 425)(SEQ ID NO: 423) NO: 423) NO: 423) VH Sequence:QMQLKQSGPGLVQPSQSLSITCTVSGFSLNDYGVHWIRQSPGKGLEWLGVIWSGGRTDYNAAFISRLSISKDNSKSQVFFKMSSLQPQDTAIYYCARWALYFLYGGSMDYWGQGTSVTVSS (SEQ ID NO: 1) VL Sequence:DVVLTQTPLSLPVSPGDQASISCRSSQSLVHSSGITYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPWTFGGGTKLEIK (SEQ ID NO: 2)

TABLE 2 Antibody 3P10 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH GYTFTDYGVI GYTFTDYG DYGVI GYTFTDY TDYGVI GYTFTDYGVI Seq.CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 48) (SEQ ID NO: 49) (SEQ ID NO: 50)(SEQ ID NO: 46) NO: 46) NO: 47) VH WINTYTGEPTYAD INTYTGEP WINTYTGEPTYADTYTG WMGWINTYTGEPT WINTYTGEPT CDR2 DLKG (SEQ ID DLKG (SEQ ID NO: 139)(SEQ ID NO: 140) (SEQ ID NO: 141) (SEQ ID NO: 138) (SEQ ID NO: 137) NO:137) VH RYGPEDIDY ARRYGPEDIDY RYGPEDIDY YGPEDID ARRYGPEDID RYGPEDIDYCDR3 (SEQ ID (SEQ ID (SEQ ID NO: 225) (SEQ ID NO: 227) (SEQ ID NO: 228)(SEQ ID NO: 225) NO: 225) NO: 226) VL CDR VL RASESVDNYGISF ESVDNYGISFRASESVDNYGISF SESVDNYGISF DNYGISFMSWF RASESVDNYGISF Seq. CDR1 MS (SEQ IDMS (SEQ ID NO: 303) (SEQ ID NO: 304) MS (SEQ ID NO: 302) (SEQ ID NO:301) (SEQ ID NO: 301) NO: 301) VL AASHQGS AAS AASHQGS AAS LLIYAASHQGAASHQGS CDR2 (SEQ ID (SEQ ID (SEQ ID NO: 376) (SEQ ID NO: 377) (SEQ IDNO: 378) (SEQ ID NO: 376) NO: 376) NO: 377) VL LQSKEVPWT LQSKEVPWTLQSKEVPWT SKEVPW LQSKEVPW LQSKEVPWT CDR3 (SEQ ID (SEQ ID (SEQ ID NO:426) (SEQ ID NO: 427) (SEQ ID NO: 428) (SEQ ID NO: 426) NO: 426) NO:426) VH Sequence:QIQLVQSGPELKKPGETVKISCKASGYTFTDYGVIWVKQAPGKALKWMGWINTYTGEPTYADDLKGRFAFSLETSASSASLQINNLKNEDTATYFCARRYGPEDIDYWGQGTTLTVSS (SEQ ID NO: 3) VL Sequence:DIVLTQSPVSLAVSLGQRATISCRASESVDNYGISFMSWFQQKPGQPPKLLIYAASHQGSGVPARFSGSGSGTDFSLNIHPMEEDDSAMYFCLQSKEVPWTFGGGTKLEIK (SEQ ID NO: 4)

TABLE 3 Antibody 12A3 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH CDR1 GYPFTIYGMN GYPFTIYG IYGMN GYPFTIY TIYGMN GYPFTIYGMNSeq. (SEQ ID (SEQ ID (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 55)(SEQ ID NO: 51) NO: 51) NO: 52) VH CDR2 WINTYSGVPTYAD INTYSGVPWINTYSGVPTYAD TYSG WMGWINTYSGVPT WINTYSGVPT DFKG (SEQ ID DFKG (SEQ IDNO: 144) (SEQ ID NO: 145) (SEQ ID NO: 146) (SEQ ID NO: 143) (SEQ ID NO:142) NO: 142) VH CDR3 ATGNY ASATGNY ATGNY TGN ASATGN ATGNY (SEQ ID (SEQID (SEQ ID NO: 229) (SEQ ID NO: 231) (SEQ ID NO: 232) (SEQ ID NO: 229)NO: 229) NO: 230) VL CDR VL CDR1 RASQDIGSSLN QDIGSS RASQDIGSSLN SQDIGSSGSSLNWL RASQDIGSSLN Seq. (SEQ ID (SEQ ID (SEQ ID NO: 305) (SEQ ID NO:307) (SEQ ID NO: 308) (SEQ ID NO: 305) NO: 305) NO: 306) VL CDR2 ATSSLDSATS ATSSLDS ATS RLIYATSSLD ATSSLDS (SEQ ID (SEQ ID (SEQ ID NO: 379) (SEQID NO: 380) (SEQ ID NO: 381) (SEQ ID NO: 379) NO: 379) NO: 380) VL CDR3LQYASSPYT LQYASSPYT LQYASSPYT YASSPY LQYASSPY LQYASSPYT (SEQ ID (SEQ ID(SEQ ID NO: 429) (SEQ ID NO: 430) (SEQ ID NO: 431) (SEQ ID NO: 429) NO:429) NO: 429) VH Sequence:QIQLVQSGPELKKPGETVKISCKASGYPFTIYGMNWVEQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKDEDTATYFCASATGNYWGQGTTLTVSS (SEQ ID NO: 5) VL Sequence:DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPYTFGGGTKVEIK (SEQ ID NO: 6)

TABLE 4 Antibody 5F12 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH GYTFTDYYIN GYTFTDYY DYYIN GYTFTDY TDYYIN GYTFTDYYIN Seq.CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 58) (SEQ ID NO: 49) (SEQ ID NO: 59)(SEQ ID NO: 56) NO: 56) NO: 57) VH RIYPGNGNTYHNE IYPGNGNT RIYPGNGNTYHNEPGNG WIARIYPGNGNTY RIYPGNGNTY CDR2 KFKG (SEQ ID KFKG (SEQ ID NO: 149)(SEQ ID NO: 150) (SEQ ID NO: 151) (SEQ ID NO: 148) (SEQ ID NO: 147) NO:147) VH EGLYYDYDRYFDY AREGLYYDYDRYF EGLYYDYDRYFDY GLYYDYDRYFDAREGLYYDYDRYFD EGLYYDYDRYFDY CDR3 (SEQ ID DY (SEQ ID NO: 233) (SEQ IDNO: 235) (SEQ ID NO: 236) (SEQ ID NO: 233) NO: 233) (SEQ ID NO: 234) VLCDR VL RASESVDTYGNSF ESVDTYGNSF RASESVDTYGNSF SESVDTYGNSF DTYGNSFMHWYRASESVDTYGNSF Seq. CDR1 MH (SEQ ID MH (SEQ ID NO: 311) (SEQ ID NO: 312)MH (SEQ ID NO: 310) (SEQ ID NO: 309) (SEQ ID NO: 309) NO: 309) VLLASNLES LAS LASNLES LAS LLIYLASNLE LASNLES CDR2 (SEQ ID (SEQ ID (SEQ IDNO: 382) (SEQ ID NO: 383) (SEQ ID NO: 384) (SEQ ID NO: 382) NO: 382) NO:383) VL HQNNEDPPA HQNNEDPPA HQNNEDPPA NNEDPP HQNNEDPP HQNNEDPPA CDR3(SEQ ID (SEQ ID (SEQ ID NO: 432) (SEQ ID NO: 433) (SEQ ID NO: 434) (SEQID NO: 432) NO: 432) NO: 432) VH Sequence:QVQLKQSGTELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPGNGNTYHNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCAREGLYYDYDRYFDYWGQGTALTVSS (SEQ ID NO: 7) VL Sequence:NIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDPPAFGGGTKLEIK (SEQ ID NO: 8)

TABLE 5 Antibody 5A20 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH GYTFTDYWIE GYTFTDYW DYWIE GYTFTDY TDYWIE GYTFTDYWIE Seq.CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 62) (SEQ ID NO: 49) (SEQ ID NO: 63)(SEQ ID NO: 60) NO: 60) NO: 61) VH EILLGSDSIHFNEK ILLGSDSIEILLGSDSIHFNEK LGSD WIGEILLGSDSIH EILLGSDSIH CDR2 FKG (SEQ ID FKG (SEQID NO: 154) (SEQ ID NO: 155) (SEQ ID NO: 156) (SEQ ID NO: 153) (SEQ IDNO: 152) NO: 152) VH QDWNWYFDV VRQDWNWYFDV QDWNWYFDV DWNWYFD VRQDWNWYFDQDWNWYFDV CDR3 (SEQ ID (SEQ ID (SEQ ID NO: 237) (SEQ ID NO: 239) (SEQ IDNO: 240) (SEQ ID NO: 237) NO: 237) NO: 238) VL CDR VL KSSQSLLDFDGKTQSLLDFDGKTY KSSQSLLDFDGKT SQSLLDFDGKTY LDFDGKTYLNWY KSSQSLLDFDGKT Seq.CDR1 YLN (SEQ ID YLN (SEQ ID NO: 315) (SEQ ID NO: 316) YLN (SEQ ID NO:314) (SEQ ID NO: 313) (SEQ ID NO: 313) NO: 313) VL LVSKLDS LVS LVSKLDSLVS RLFYLVSKLD LVSKLDS CDR2 (SEQ ID (SEQ ID (SEQ ID NO: 385) (SEQ ID NO:386) (SEQ ID NO: 387) (SEQ ID NO: 385) NO: 385) NO: 386) VL WQGTHFPRTWQGTHFPRT WQGTHFPRT GTHFPR WQGTHFPR WQGTHFPRT CDR3 (SEQ ID (SEQ ID (SEQID NO: 435) (SEQ ID NO: 436) (SEQ ID NO: 437) (SEQ ID NO: 435) NO: 435)NO: 435) VH Sequence:QVQLQQSGPELMKPGASVILSCKAIGYTFTDYWIEWVKERPGHGLEWIGEILLGSDSIHFNEKFKGKATISADTSSNTAYMQLSSLTTEDSAIYYCVRQDWNWYFDVWGTGTTVTVSS (SEQ ID NO: 9) VL Sequence:DVVMTQTPLTLSVTIGHPASISCKSSQSLLDFDGKTYLNWLFQRPGQSPKRLFYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 10)

TABLE 6 Antibody 8D8 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH VH CDR1 GFSLSRYSVH GFSLSRYS RYSVH GFSLSRY SRYSVH GFSLSRYSVH CDR(SEQ ID (SEQ ID (SEQ ID NO: 66) (SEQ ID NO: 67) (SEQ ID NO: 68) (SEQ IDNO: 64) Seq. NO: 64) NO: 65) VH CDR2 MIWGFGSTDYNS IWGFGST MIWGFGSTDYNSGFG WLGMIWGFGSTD MIWGFGSTD ALKS (SEQ ID ALKS (SEQ ID NO: 158) (SEQ IDNO: 159) (SEQ ID NO: 160) (SEQ ID NO: 220) (SEQ ID NO: 157) NO: 157) VHCDR3 IHTTAGSY ARIHTTAGSY IHTTAGSY HTTAGS ARIHTTAGS IHTTAGSY (SEQ ID (SEQID (SEQ ID NO: 241) (SEQ ID NO: 243) (SEQ ID NO: 244) (SEQ ID NO: 241)NO: 241) NO: 242) VL VL CDR1 KASQNVGTNVA QNVGTN KASQNVGTNVA SQNVGTNGTNVAWY KASQNVGTNVA CDR (SEQ ID (SEQ ID (SEQ ID NO: 317) (SEQ ID NO:319) (SEQ ID NO: 320) (SEQ ID NO: 317) Seq. NO: 317) NO: 318) VL CDR2STSYRYS STS STSYRYS STS ALVYSTSYRY STSYRYS (SEQ ID (SEQ ID (SEQ ID NO:388) (SEQ ID NO: 389) (SEQ ID NO: 390) (SEQ ID NO: 388) NO: 388) NO:389) VL CDR3 HQYNSYPLT HQYNSYPLT HQYNSYPLT YNSYPL HQYNSYPL HQYNSYPLT(SEQ ID (SEQ ID (SEQ ID NO: 438) (SEQ ID NO: 439) (SEQ ID NO: 440) (SEQID NO: 438) NO: 438) NO: 438) VH Sequence:QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGFGSTDYNSALKSRLSITKDNSKSQFFLKMNSLQTDDTAMYYCARIHTTAGSYWGQGTLVTVSA (SEQ ID NO: 11) VL Sequence:DIVMTQSQKFMSTSIGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALVYSTSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQYNSYPLTFGAGTKLELK (SEQ ID NO: 12)

TABLE 7 Antibody 17J16 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH VH CDR1 GYTFTDYWIH GYTFTDYW DYWIH GYTFTDY TDYWIHGYTFTDYWIH CDR (SEQ ID (SEQ ID (SEQ ID NO: 70) (SEQ ID NO: 49) (SEQ IDNO: 71) (SEQ ID NO: 69) Seq. NO: 69) NO: 61) VH CDR2 YINPNSNYAEYNQINPNSNYA YINPNSNYAEYNQ PNSN WIGYINPNSNYAE YINPNSNYAE KFKV (SEQ ID KFKV(SEQ ID NO: 163) (SEQ ID NO: 164) (SEQ ID NO: 165) (SEQ ID NO: 162) (SEQID NO: 161) NO: 161) VH CDR3 FDWNWYFHV ARFDWNWYFHV FDWNWYFHV DWNWYFHARFDWNWYFH FDWNWYFHV (SEQ ID (SEQ ID (SEQ ID NO: 245) (SEQ ID NO: 247)(SEQ ID NO: 248) (SEQ ID NO: 245) NO: 245) NO: 246) VL VL CDR1KSSQSLSDSDGKT QSLSDSDGKTY KSSQSLSDSDGKT SQSLSDSDGKTY SDSDGKTYLNWLKSSQSLSDSDGKT CDR YLN (SEQ ID YLN (SEQ ID NO: 323) (SEQ ID NO: 324) YLNSeq. (SEQ ID NO: 322) (SEQ ID NO: 321) (SEQ ID NO: 321) NO: 321) VL CDR2LVSRLGS LVS LVSRLGS LVS RLIYLVSRLG LVSRLGS (SEQ ID (SEQ ID (SEQ ID NO:391) (SEQ ID NO: 386) (SEQ ID NO: 392) (SEQ ID NO: 391) NO: 391) NO:386) VL CDR3 WQGTHFPQT WQGTHFPQT WQGTHFPQT GTHFPQ WQGTHFPQ WQGTHFPQT(SEQ ID (SEQ ID (SEQ ID NO: 441) (SEQ ID NO: 442) (SEQ ID NO: 443) (SEQID NO: 441) NO: 441) NO: 441) VH Sequence:QVQLQQSGAELAKPGASVKMSCKTSGYTFTDYWIHWVKQRPGQGLEWIGYINPNSNYAEYNQKFKVKATLTADKSSSTAYLQLSRLTSEDSAVYYCARFDWNWYFHVWGAGSTVTVSS (SEQ ID NO: 13) VL Sequence:DVALTQIPLTLSVTVGQPASISCKSSQSLSDSDGKTYLNWLLQKPGQSPKRLIYLVSRLGSGVPDRFTGSGSGADFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK (SEQ ID NO: 14)

TABLE 8 Antibody 25M22 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH GYTFTSYWVN GYTFTSYW SYWVN GYTFTSY TSYWVNGYTFTSYWVN Seq. CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 74) (SEQ ID NO: 75)(SEQ ID NO: 76) (SEQ ID NO: 72) NO: 72) NO: 73) VH RIYPGDGDTNYNGIYPGDGDT RIYPGDGDTNYNG PGDG WIGRIYPGDGDTN RIYPGDGDTN CDR2 KFKG (SEQ IDKFKG (SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170) (SEQ ID NO: 167)(SEQ ID NO: 166) NO: 166) VH AYLLRLRRTGYYA ARAYLLRLRRTGY AYLLRLRRTGYYAYLLRLRRTGYYAMD ARAYLLRLRRTGY AYLLRLRRTGYYA CDR3 MDY YAMDY MDY (SEQ IDNO: 251) YAMD MDY (SEQ ID (SEQ ID (SEQ ID NO: 249) (SEQ ID NO: 252) (SEQID NO: 249) NO: 249) NO: 250) VL CDR VL KSTKSLLNSDEFT KSLLNSDEFTYKSTKSLLNSDEFT TKSLLNSDEFTY LNSDEFTYLDWY KSTKSLLNSDEFT Seq. CDR1 YLD (SEQID YLD (SEQ ID NO: 327) (SEQ ID NO: 328) YLD (SEQ ID NO: 326) (SEQ IDNO: 325) (SEQ ID NO: 325) NO: 325) VL LVSNRFS LVS LVSNRFS LVS LLIFLVSNRFLVSNRFS CDR2 (SEQ ID (SEQ ID (SEQ ID NO: 393) (SEQ ID NO: 386) (SEQ IDNO: 394) (SEQ ID NO: 393) NO: 393) NO: 386) VL FQSNYLPYT FQSNYLPYTFQSNYLPYT SNYLPY FQSNYLPY FQSNYLPYT CDR3 (SEQ ID (SEQ ID (SEQ ID NO:444) (SEQ ID NO: 445) (SEQ ID NO: 446) (SEQ ID NO: 444) NO: 444) NO:444) VH Sequence:QVQLQQSGPDLVKPGASVKISCKASGYTFTSYWVNWMKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARAYLLRLRRTGYYAMDYWGQGTSVTVSS (SEQ ID NO: 15) VL Sequence:DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDEFTYLDWYLQKPGQSPQLLIFLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPYTFGGGTKLEIK (SEQ ID NO: 16)

TABLE 9 Antibody 2B8 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH GYTFTTYGMS GYTFTTYG TYGMS GYTFTTY TTYGMS GYTFTTYGMS Seq.CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 79) (SEQ ID NO: 80) (SEQ ID NO: 81)(SEQ ID NO: 77) NO: 77) NO: 78) VH WINTYSGVPTFVD INTYSGVP WINTYSGVPTFVDTYSG WMGWINTYSGVPT WINTYSGVPT CDR2 DFRG (SEQ ID DFRG (SEQ ID NO: 144)(SEQ ID NO: 145) (SEQ ID NO: 146) (SEQ ID NO: 143) (SEQ ID NO: 171) NO:171) VH RSSYYPYWYFDV ARRSSYYPYWYF RSSYYPYWYFDV SSYYPYWYFD ARRSSYYPYWYFDRSSYYPYWYFDV CDR3 (SEQ ID DV (SEQ ID NO: 253) (SEQ ID NO: 255) (SEQ IDNO: 256) (SEQ ID NO: 253) NO: 253) (SEQ ID NO: 254) VL CDR VLRPSENIYSYLT ENIYSY RPSENIYSYLT SENIYSY YSYLTWF RPSENIYSYLT Seq. CDR1(SEQ ID (SEQ ID (SEQ ID NO: 329) (SEQ ID NO: 331) (SEQ ID NO: 332) (SEQID NO: 329) NO: 329) NO: 330) VL NAQTLAE NAQ NAQTLAE NAQ LLVYNAQTLANAQTLAE CDR2 (SEQ ID (SEQ ID (SEQ ID NO: 395) (SEQ ID NO: 396) (SEQ IDNO: 397) (SEQ ID NO: 395) NO: 395) NO: 396) VL QHYYGYPFT QHYYGYPFTQHYYGYPFT YYGYPF QHYYGYPF QHYYGYPFT CDR3 (SEQ ID (SEQ ID (SEQ ID NO:447) (SEQ ID NO: 448) (SEQ ID NO: 449) (SEQ ID NO: 447) NO: 447) NO:447) VH Sequence:QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKIFKWMGWINTYSGVPTFVDDFRGRFAFSLETSASTAYLQIGNLKNEDTATYFCARRSSYYPYWYFDVWGTGTTVTVSS (SEQ ID NO: 17) VL Sequence:DIQMTQSPASLSASVGETVTITCRPSENIYSYLTWFQQEQGKSPQLLVYNAQTLAEGVPSRFSGSGSGTHFSLKINSLQPEDFGTYYCQHYYGYPFTFGSGTKLEIK (SEQ ID NO: 18)

TABLE 10 Antibody 22N5 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH CDR1 GYTFTDYSMH GYTFTDYS DYSMH GYTFTDY TDYSMHGYTFTDYSMH Seq. (SEQ ID (SEQ ID (SEQ ID NO: 84) (SEQ ID NO: 49) (SEQ IDNO: 85) (SEQ ID NO: 82) NO: 82) NO: 83) VH CDR2 WINTETGEPTYAD INTETGEPWINTETGEPTYAD TETG WMGWINTETGEPT WINTETGEPT DFKG (SEQ ID DFKG (SEQ IDNO: 174) (SEQ ID NO: 175) (SEQ ID NO: 176) (SEQ ID NO: 173) (SEQ ID NO:172) NO: 172) VH CDR3 GTLNY VKGTLNY GTLNY TLN VKGTLN GTLNY (SEQ ID (SEQID (SEQ ID NO: 257) (SEQ ID NO: 259) (SEQ ID NO: 260) (SEQ ID NO: 257)NO: 257) NO: 258) VL CDR VL CDR1 KASQDIKSYLN QDIKSY KASQDIKSYLN SQDIKSYKSYLNWF KASQDIKSYLN Seq. (SEQ ID (SEQ ID (SEQ ID NO: 333) (SEQ ID NO:335) (SEQ ID NO: 336) (SEQ ID NO: 333) NO: 333) NO: 334) VL CDR2 RTKRLVDRTK RTKRLVD RTK TLIYRTKRLV RTKRLVD (SEQ ID (SEQ ID (SEQ ID NO: 398) (SEQID NO: 399) (SEQ ID NO: 400) (SEQ ID NO: 398) NO: 398) NO: 399) VL CDR3LQYVEFPLT LQYVEFPLT LQYVEFPLT YVEFPL LQYVEFPL LQYVEFPLT (SEQ ID (SEQ ID(SEQ ID NO: 450) (SEQ ID NO: 451) (SEQ ID NO: 452) (SEQ ID NO: 450) NO:450) NO: 450) VH Sequence:QNQLVQSGPELKKPGEIVKISCKTSGYTFTDYSMHWVKKTPGKGFKWMGWINTETGEPTYADDFKGRFAFSLETSANTAHLQITNLKNEDTATYFCVKGTLNYWGQGTTLTVSS (SEQ ID NO: 19) VL Sequence:DIKMTQSPSSMYASLGERVTITCKASQDIKSYLNWFQQKPGKSPKTLIYRTKRLVDGVPSRFSGSGSGQDYSLTVSSLEYDDVGIYYCLQYVEFPLTFGDGTKLELK (SEQ ID NO: 20)

TABLE 11 Antibody 2I23 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH GYSFTSYNID GYSFTSYN SYNID GYSFTSY TSYNIDGYSFTSYNID Seq. CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 88) (SEQ ID NO: 89)(SEQ ID NO: 90) (SEQ ID NO: 86) NO: 86) NO: 87) VH WIFPGDGST IFPGDGSTWIFPGDGSTKYNE PGDG WIGWIFPGDGSTK WIFPGDGSTK CDR2 (SEQ ID (SEQ ID KFKG(SEQ ID NO: 168) (SEQ ID NO: 180) (SEQ ID NO: 181) NO: 177) NO: 178)(SEQ ID NO: 179) VH SGIYYGSHFVY ARSGIYYGSHFVY SGIYYGSHFVY GIYYGSHFVARSGIYYGSHFV SGIYYGSHFVY CDR3 (SEQ ID (SEQ ID (SEQ ID NO: 261) (SEQ IDNO: 263) (SEQ ID NO: 264) (SEQ ID NO: 261) NO: 261) NO: 262) VL CDR VLRSSQSLLDSDGKT QSLLDSDGKTY RSSQSLLDSDGKT SQSLLDSDGKTY LDSDGKTYLNWLRSSQSLLDSDGKT Seq. CDR1 YLN (SEQ ID YLN (SEQ ID NO: 339) (SEQ ID NO:340) YLN (SEQ ID NO: 338) (SEQ ID NO: 337) (SEQ ID NO: 337) NO: 337) VLLVSKVDS LVS LVSKVDS LVS RLIYLVSKVD LVSKVDS CDR2 (SEQ ID (SEQ ID (SEQ IDNO: 401) (SEQ ID NO: 386) (SEQ ID NO: 402) (SEQ ID NO: 401) NO: 401) NO:386) VL WQGTHFPLT WQGTHFPLT WQGTHFPLT GTHFPL WQGTHFPL WQGTHFPLT CDR3(SEQ ID (SEQ ID (SEQ ID NO: 453) (SEQ ID NO: 454) (SEQ ID NO: 455) (SEQID NO: 453) NO: 453) NO: 453) VH Sequence:QAQLQQSGAELVKPGASVKLSCKASGYSFTSYNIDWVRQRPEQGLEWIGWIFPGDGSTKYNEKFKGQATLTTDKSSSTTYIHLSRLTSEDSAVYFCARSGIYYGSHFVYWGQGTLVTVSA (SEQ ID NO: 21) VL Sequence:DVVMTQTPLTLSVTIGQSASISCRSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCWQGTHFPLTFGAGTKLELK (SEQ ID NO: 22)

TABLE 12 Antibody 6N16 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH CDR1 GYTFTSYNIN GYTFTSYN SYNIN GYTFTSY TSYNINGYTFTSYNIN Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 75) (SEQ ID NO: 94)(SEQ ID NO: 91) NO: 91) NO: 92) NO: 93) VH CDR2 WIFPGDDSIKYNE IFPGDDSIWIFPGDDSIKYNE PGDD WIGWIFPGDDSIK WIFPGDDSIK NFRG (SEQ ID NFRG (SEQ IDNO: 184) (SEQ ID NO: 185) (SEQ ID NO: 186) (SEQ ID NO: 183) (SEQ ID NO:182) NO: 182) VH CDR3 SGIFYGNNFAY ARSGIFYGNNFAY SGIFYGNNFAY GIFYGNNFAARSGIFYGNNFA SGIFYGNNFAY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 267) (SEQID NO: 268) (SEQ ID NO: 265) NO: 265) NO: 266) NO: 265) VL CDR VL CDR1KSSQSLLDGDGET QSLLDGDGETY KSSQSLLDGDGET SQSLLDGDGETY LDGDGETYLSWLKSSQSLLDGDGET Seq. YLS (SEQ ID YLS (SEQ ID NO: 343) (SEQ ID NO: 344) YLS(SEQ ID NO: 342) (SEQ ID (SEQ ID NO: 341) NO: 341) NO: 341) VL CDR2LVSKLDS LVS LVSKLDS LVS RLIYLVSKLD LVSKLDS (SEQ ID (SEQ ID (SEQ ID (SEQID NO: 386) (SEQ ID NO: 403) (SEQ ID NO: 385) NO: 385) NO: 386) NO: 385)VL CDR3 CQSTHFPLT CQSTHFPLT CQSTHFPLT STHFPL CQSTHFPL CQSTHFPLT (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 457) (SEQ ID NO: 458) (SEQ ID NO: 456) NO:456) NO: 456) NO: 456) VH Sequence:QVQLQQSGSELVKPGTSMKLSCKASGYTFTSYNINWVRLRPEQGLEWIGWIFPGDDSIKYNENFRGKATLTTDKSSSTAYMHLSRLTSDDSAVYFCARSGIFYGNNFAYWGQGTLVTVSA (SEQ ID NO: 23) VL Sequence:DVVMTQAPLILSVTIGQPASISCKSSQSLLDGDGETYLSWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCCQSTHFPLTFGAGTKLELK (SEQ ID NO: 24)

TABLE 13 Antibody 1B3 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH CDR1 GFTFTGYNIN GFTFTGYN GYNIN GFTFTGY TGYNIN GFTFTGYNINSeq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 98) (SEQ ID NO: 99) (SEQ ID NO:95) NO: 95) NO: 96) NO: 97) VH CDR2 WIFPGDDNAKYNE IFPGDDNA WIFPGDDNAKYNEPGDD WIGWIFPGDDNAK WIFPGDDNAK KFKG (SEQ ID KFKG (SEQ ID NO: 184) (SEQ IDNO: 189) (SEQ ID NO: 190) (SEQ ID NO: 188) (SEQ ID NO: 187) NO: 187) VHCDR3 TPVLSNYFDY ARTPVLSNYFDY TPVLSNYFDY PVLSNYFD ARTPVLSNYFD TPVLSNYFDY(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 271) (SEQ ID NO: 272) (SEQ ID NO:269) NO: 269) NO: 270) NO: 269) VL CDR VL CDR1 KASQDISKYIS QDISKYKASQDISKYIS SQDISKY SKYISWY KASQDISKYIS Seq. (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 347) (SEQ ID NO: 348) (SEQ ID NO: 345) NO: 345) NO: 346) NO:345) VL CDR2 YTSTLQP YTS YTSTLQP YTS LLIHYTSTLQ YTSTLQP (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 405) (SEQ ID NO: 406) (SEQ ID NO: 404) NO: 404) NO:405) NO: 404) VL CDR3 LQYDNLYT LQYDNLYT LQYDNLYT YDNLY LQYDNLY LQYDNLYT(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 460) (SEQ ID NO: 461) (SEQ ID NO:459) NO: 459) NO: 459) NO: 459) VH Sequence:QVHLQQPGAELVKPGASVKLSCKASGFTFTGYNINWVRLRPEQGLEWIGWIFPGDDNAKYNEKFKGKATLTTDKSSNTAYMQLSRLTSEDSAVYFCARTPVLSNYFDYWGQGTTLTVSS (SEQ ID NO: 25) VL Sequence:DIQMTQSPSSLSASLGGKVTITCKASQDISKYISWYQHKPGKSPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK (SEQ ID NO: 26)

TABLE 14 Antibody 19K19 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH CDR1 GYAFTSYWMN GYAFTSYW SYWMN GYAFTSY TSYWMNGYAFTSYWMN Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 103) (SEQ ID NO:104) (SEQ ID NO: 100) NO: 100) NO: 101) NO: 102) VH CDR2 RIYPGDGDTNYNGIYPGDGDT RIYPGDGDTNYNG PGDG WIGRIYPGDGDTN RIYPGDGDTN KFKG (SEQ ID KFKG(SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170) (SEQ ID NO: 167) (SEQID NO: 166) NO: 166) VH CDR3 AYLLRLRRTGYYA ARAYLLRLRRTGY AYLLRLRRTGYYAYLLRLRRTGYYAMD ARAYLLRLRRTGY AYLLRLRRTGYYA MDY YAMDY MDY (SEQ ID NO:251) YAMD MDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 252) (SEQ ID NO: 249)NO: 249) NO: 250) NO: 249) VL CDR VL CDR1 KSTKSLLNSDEFT KSLLNSDEFTYKSTKSLLNSDEFT TKSLLNSDEFTY LNSDEFTYLDWY KSTKSLLNSDEFT Seq. YLD (SEQ IDYLD (SEQ ID NO: 327) (SEQ ID NO: 328) YLD (SEQ ID NO: 326) (SEQ ID (SEQID NO: 325) NO: 325) NO: 325) VL CDR2 LVSNRFS LVS LVSNRFS LVS LLIYLVSNRFLVSNRFS (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 386) (SEQ ID NO: 407) (SEQID NO: 393) NO: 393) NO: 386) NO: 393) VL CDR3 FQSNYLPYT FQSNYLPYTFQSNYLPYT SNYLPY FQSNYLPY FQSNYLPYT (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:445) (SEQ ID NO: 446) (SEQ ID NO: 444) NO: 444) NO: 444) NO: 444) VHSequence:QVQLQQSGPDLVKPGASVKISCKASGYAFTSYWMNWVKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARAYLLRLRRTGYYAMDYWGQGTSVTVSS (SEQ ID NO: 27) VL Sequence:DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDEFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPYTFGGGTKLEIK (SEQ ID NO: 28)

TABLE 15 Antibody 2B3 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH CDR1 GFTFSDYFMF GFTFSDYF DYFMF GFTFSDY SDYFMF GFTFSDYFMFSeq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 108) (SEQ ID NO: 109) (SEQ IDNO: 105) NO: 105) NO: 106) NO: 107) VH CDR2 YISNDGDSTYYPD ISNDGDSTYISNDGDSTYYPD NDGD WVAYISNDGDSTY YISNDGDSTY TVQG (SEQ ID TVQG (SEQ IDNO: 193) (SEQ ID NO: 194) (SEQ ID NO: 195) (SEQ ID NO: 192) (SEQ ID NO:191) NO: 191) VH CDR3 QGAQATLDY TRQGAQATLDY QGAQATLDY GAQATLD TRQGAQATLDQGAQATLDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 275) (SEQ ID NO: 276) (SEQID NO: 273) NO: 273) NO: 274) NO: 273) VL CDR VL CDR1 SASSSVFYMH SSVFYSASSSVFYMH SSSVFY FYMHWY SASSSVFYMH Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 351) (SEQ ID NO: 352) (SEQ ID NO: 349) NO: 349) NO: 350) NO: 349) VLCDR2 STSNLAS STS STSNLAS STS LLIYSTSNLA STSNLAS (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 389) (SEQ ID NO: 409) (SEQ ID NO: 408) NO: 408) NO: 389) NO:408) VL CDR3 HQWSST HQWSST HQWSST WSS HQWSS HQWSST (SEQ ID (SEQ ID (SEQID (SEQ ID NO: 463) (SEQ ID NO: 464) (SEQ ID NO: 462) NO: 462) NO: 462)NO: 462) VH Sequence:EVKLVESGGGLVQPGGSLKLSCAASGFTFSDYFMFWVRQTPEKRLEWVAYISNDGDSTYYPDTVQGRFTISRDNAKNTLYLQMSRLRSEDTAMYYCTRQGAQATLDYWGQGTTLTVSS (SEQ ID NO: 29) VL Sequence:QIVLTQSPAIMSASLGEEITLTCSASSSVFYMHWYQQKSGTSPKLLIYSTSNLASGIPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSTFGGGTKLEIK (SEQ ID NO: 30)

TABLE 16 Antibody 8C10 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH CDR1 GYTFANYGLT GYTFANYG NYGLT GYTFANY ANYGLTGYTFANYGLT Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 113) (SEQ ID NO:114) (SEQ ID NO: 110) NO: 110) NO: 111) NO: 112) VH CDR2 EIYPGSGHTHYNEIYPGSGHT EIYPGSGHTHYNE PGSG WIGEIYPGSGHTH EIYPGSGHTH DFKG (SEQ ID DFKG(SEQ ID NO: 198) (SEQ ID NO: 199) (SEQ ID NO: 200) (SEQ ID NO: 197) (SEQID NO: 196) NO: 196) VH CDR3 RIQLLLPVGGFVY ARRIQLLLPVGGF RIQLLLPVGGFVYIQLLLPVGGFV ARRIQLLLPVGGFV RIQLLLPVGGFVY (SEQ ID VY (SEQ ID (SEQ ID NO:279) (SEQ ID NO: 280) (SEQ ID NO: 277) NO: 277) (SEQ ID NO: 277) NO:278) VL CDR VL CDR1 RASQSISNNLH QSISNN RASQSISNNLH SQSISNN SNNLHWYRASQSISNNLH Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 355) (SEQ ID NO:356) (SEQ ID NO: 353) NO: 353) NO: 354) NO: 353) VL CDR2 YASQSIS YASYASQSIS YAS LLIKYASQSI YASQSIS (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 411)(SEQ ID NO: 412) (SEQ ID NO: 410) NO: 410) NO: 411) NO: 410) VL CDR3QQSNSWPHT QQSNSWPHT QQSNSWPHT SNSWPH QQSNSWPH QQSNSWPHT (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 466) (SEQ ID NO: 467) (SEQ ID NO: 465) NO: 465) NO:465) NO: 465) VH Sequence:QVQLQQSGVELARPGAAVKLSCKASGYTFANYGLTWVKQRTGQGLEWIGEIYPGSGHTHYNEDFKGKATLTADRSSSTAYMELRSLTSEDSAVYFCARRIQLLLPVGGFVYWGQGTLVTVSA (SEQ ID NO: 31) VL Sequence:DFVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSNSWPHTFGGGTKLEIK (SEQ ID NO: 32)

TABLE 17 Antibody 2A9 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH CDR1 GFTFSTYAMS GFTFSTYA TYAMS GFTFSTY STYAMS GFTFSTYAMSSeq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 118) (SEQ ID NO: 119) (SEQ IDNO: 115) NO: 115) NO: 116) NO: 117) VH CDR2 SITSGGTTYYTDS ITSGGTTSITSGGTTYYTDS SGG WVASITSGGTTY SITSGGTTY VKG (SEQ ID VKG (SEQ ID NO:134) (SEQ ID NO: 203) (SEQ ID NO: 204) (SEQ ID NO: 202) (SEQ ID NO: 201)NO: 201) VH CDR3 DGNFYYYGMDY ARDGNFYYYGMDY DGNFYYYGMDY GNFYYYGMDARDGNFYYYGMD DGNFYYYGMDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 283) (SEQID NO: 284) (SEQ ID NO: 281) NO: 281) NO: 282) NO: 281) VL CDR VL CDR1KASQNVGTAVA QNVGTA KASQNVGTAVA SQNVGTA GTAVAWY KASQNVGTAVA Seq. (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 359) (SEQ ID NO: 360) (SEQ ID NO: 357) NO:357) NO: 358) NO: 357) VL CDR2 SASNRFT SAS SASNRFT SAS ILIYSASNRFSASNRFT (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 414) (SEQ ID NO: 415) (SEQID NO: 413) NO: 413) NO: 414) NO: 413) VL CDR3 QQYSSYFT QQYSSYFTQQYSSYFT YSSYF QQYSSYF QQYSSYFT (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 469)(SEQ ID NO: 470) (SEQ ID NO: 468) NO: 468) NO: 468) NO: 468) VHSequence:EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASITSGGTTYYTDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDGNFYYYGMDYWGQGTSVTVSS (SEQ ID NO: 33) VL Sequence:DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSPKILIYSASNRFTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYFTFGGGTKLELK (SEQ ID NO: 34)

TABLE 18 Antibody 24G2 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH CDR1 GYTFTTYWMH GYTFTTYW TYWMH GYTFTTY TTYWMHGYTFTTYWMH Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 80) (SEQ ID NO: 123)(SEQ ID NO: 120) NO: 120) NO: 121) NO: 122) VH CDR2 MIHPNSGSSNYNEIHPNSGSS MIHPNSGSSNYNE PNSG WIGMIHPNSGSSN MIHPNSGSSN KFKN (SEQ ID KFKN(SEQ ID NO: 207) (SEQ ID NO: 208) (SEQ ID NO: 209) (SEQ ID NO: 206) (SEQID NO: 205) NO: 205) VH CDR3 SDYGFIPYFDY ARSDYGFIPYFDY SDYGFIPYFDYDYGFIPYFD ARSDYGFIPYFD SDYGFIPYFDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:287) (SEQ ID NO: 288) (SEQ ID NO: 285) NO: 285) NO: 286) NO: 285) VL CDRVL CDR1 RASQSIGTSIH QSIGTS RASQSIGTSIH SQSIGTS GTSIHWY RASQSIGTSIH Seq.(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 363) (SEQ ID NO: 364) (SEQ ID NO:361) NO: 361) NO: 362) NO: 361) VL CDR2 YASESIS YAS YASESIS YASLLIKYASESI YASESIS (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 411) (SEQ ID NO:417) (SEQ ID NO: 416) NO: 416) NO: 411) NO: 416) VL CDR3 QQSNSWPTFTQQSNSWPTFT QQSNSWPTFT SNSWPTF QQSNSWPTF QQSNSWPTFT (SEQ ID (SEQ ID (SEQID (SEQ ID NO: 472) (SEQ ID NO: 473) (SEQ ID NO: 471) NO: 471) NO: 471)NO: 471) VH Sequence:QVQLQQSGAELLKPGASVKLSCKASGYTFTTYWMHWVKQRPGQGLEWIGMIHPNSGSSNYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARSDYGFIPYFDYWGQGTTLTVSS (SEQ ID NO: 35) VL Sequence:DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLIINSVESEDIADYYCQQSNSWPTFTFGAGTKLELK (SEQ ID NO: 36)

TABLE 19 Antibody 6G9 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH CDR1 GYTFTSYWMQ GYTFTSYW SYWMQ GYTFTSY TSYWMQ GYTFTSYWMQSeq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 75) (SEQ ID NO: 126) (SEQ IDNO: 124) NO: 124) NO: 73) NO: 125) VH CDR2 EIDPSDSYTNYNQ IDPSDSYTEIDPSDSYTNYNQ PSDS WIGEIDPSDSYTN EIDPSDSYTN KFKG (SEQ ID KFKG (SEQ IDNO: 212) (SEQ ID NO: 213) (SEQ ID NO: 214) (SEQ ID NO: 211) (SEQ ID NO:210) NO: 210) VH CDR3 PLDRSAYYFDY ARPLDRSAYYFDY PLDRSAYYFDY LDRSAYYFDARPLDRSAYYFD PLDRSAYYFDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 291) (SEQID NO: 292) (SEQ ID NO: 289) NO: 289) NO: 290) NO: 289) VL CDR VL CDR1RASESVDFSGNSF ESVDFSGNSF RASESVDFSGNSF SESVDFSGNSF DFSGNSFMHWYRASESVDFSGNSF Seq. MH (SEQ ID MH (SEQ ID NO: 367) (SEQ ID NO: 368) MH(SEQ ID NO: 366) (SEQ ID (SEQ ID NO: 365) NO: 365) NO: 365) VL CDR2RASNLDS RAS RASNLDS RAS LLIYRASNLD RASNLDS (SEQ ID (SEQ ID (SEQ ID (SEQID NO: 419) (SEQ ID NO: 420) (SEQ ID NO: 418) NO: 418) NO: 419) NO: 418)VL CDR3 QQSNEDPYT QQSNEDPYT QQSNEDPYT SNEDPY QQSNEDPY QQSNEDPYT (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 475) (SEQ ID NO: 476) (SEQ ID NO: 474) NO:474) NO: 474) NO: 474) VH Sequence:QVQLHQPGAELVKPGASVKLSCKTSGYTFTSYWMQWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKATLTVDTSSTTAYMQLSSLTSEDSAVYYCARPLDRSAYYFDYWGQGTTLTVSS (SEQ ID NO: 37) VL Sequence:DIVLTQSPASLAVSLGQRATISCRASESVDFSGNSFMHWYQQKPGQPPKWYRASNLDSGIPARFSGVGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIE (SEQ ID NO: 38)

TABLE 20 Antibody 2B11 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH CDR1 GYSITSGYYWN GYSITSGYY SGYYWN GYSITSGY TSGYYWNGYSITSGYYWN Seq. (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 130) (SEQ ID NO:131) (SEQ ID NO: 127) NO: 127) NO: 128) NO: 129) VH CDR2 HIANDGSNYYNPFIANDGSN HIANDGSNYYNPF NDG WMGHIANDGSNY HIANDGSNY LKH (SEQ ID LKH (SEQ IDNO: 217) (SEQ ID NO: 218) (SEQ ID NO: 219) (SEQ ID NO: 216) (SEQ ID NO:215) NO: 215) VH CDR3 GGSYFDYVDY ARGGSYFDYVDY GGSYFDYVDY GSYFDYVDARGGSYFDYVD GGSYFDYVDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 295) (SEQ IDNO: 296) (SEQ ID NO: 293) NO: 293) NO: 294) NO: 293) VL CDR VL CDR1RASQDISNYLN QDISNY RASQDISNYLN SQDISNY SNYLNWY RASQDISNYLN Seq. (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 371) (SEQ ID NO: 372) (SEQ ID NO: 369) NO:369) NO: 370) NO: 369) VL CDR2 YTSRLHS YTS YTSRLHS YTS LLIYYTSRLHYTSRLHS (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 405) (SEQ ID NO: 422) (SEQID NO: 421) NO: 421) NO: 405) NO: 421) VL CDR3 QQGNTLPFT QQGNTLPFTQQGNTLPFT GNTLPF QQGNTLPF QQGNTLPFT (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:478) (SEQ ID NO: 479) (SEQ ID NO: 477) NO: 477) NO: 477) NO: 477) VHSequence:DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGHIANDGSNYYNPFLKHRVSITRDTSKNQFFLKLNSVTIQDTATYYCARGGSYFDYVDYWGQGTTLTVSS (SEQ ID NO: 39) VL Sequence:DIQMTQTTSSLSASLGDRVTINCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTITNLEQEDIATYFCQQGNTLPFTFGSGTKLEIK (SEQ ID NO: 40)

TABLE 21 Antibody 1A3 CDR Sequences Exemplary IMGT Kabat Chothia ContactAbM VH CDR VH GFTFTDYYMN GFTFTDYY DYYMN GFTFTDY TDYYMN GFTFTDYYMN Seq.CDR1 (SEQ ID (SEQ ID NO: (SEQ ID NO: 490) (SEQ ID NO: (SEQ ID NO: 491)(SEQ ID NO: 488) NO: 488) 1795) 1796) VH DIIPNNGVTSYNQ IIPNNGVTDIIPNNGVTSYNQ PNNG WIGDIIPNNGVTS DIIPNNGVTS CDR2 KFKG (SEQ ID (SEQ IDKFKG (SEQ ID (SEQ ID NO: 499) (SEQ ID NO: 500) (SEQ ID NO: 501) NO: 497)NO: 498) NO: 497) VH EWLLRGMDY AREWLLRGMDY EWLLRGMDY WLLRGMD AREWLLRGMDEWLLRGMDY CDR3 (SEQ ID (SEQ ID (SEQ ID NO: 515) (SEQ ID NO: 517) (SEQ IDNO: 518) (SEQ ID NO: 515) NO: 515) NO: 516) VL CDR VL RSSKSLLHSNGITKSLLHSNGITY RSSKSLLHSNGIT SKSLLHSNGITY LHSNGITYLYWY RSSKSLLHSNGIT Seq.CDR1 YLY (SEQ ID (SEQ ID YLY (SEQ ID NO: (SEQ ID NO: 533) (SEQ ID NO:534) YLY (SEQ ID NO: NO: 531) NO: 532) 531) 531) VL QMSNLAS QMS QMSNLASQMS LLIYQMSNLA QMSNLAS CDR2 (SEQ ID (SEQ ID (SEQ ID NO: 547) (SEQ ID NO:548) (SEQ ID NO: 549) (SEQ ID NO: 547) NO: 547) NO: 548) VL AQHLELTWTAQHLELTWT AQHLELTWT HLELTW AQHLELTW AQHLELTWT CDR3 (SEQ ID (SEQ ID (SEQID NO: 558) (SEQ ID NO: 559) (SEQ ID NO: 560) (SEQ ID NO: 558) NO: 558)NO: 558) VH Sequence:EVQLQQSGPELVKPGASVKISCKASGFTFTDYYMNWVKQSHGKSLEWIGDIIPNNGVTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREWLLRGMDYWGQGTSVTVSS (SEQ ID NO: 480) VL Sequence:DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQHLELTWTFGGGTKLEIK (SEQ ID NO: 481)

TABLE 22 Antibody P1B6 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH GYTFTDYYMN GYTFTDYY DYYMN GYTFTDY TDYYMNGYTFTDYYMN Seq. CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 490) (SEQ ID NO: 49)(SEQ ID NO: 491) (SEQ ID NO: 489) NO: 489) NO: 57) VH DINPNNGGPIYNINPNNGGP DINPNNGGPIYN PNNG WIGDINPNNGGPI DINPNNGGPI CDR2 QKFKG (SEQ ID(SEQ ID QKFKG (SEQ ID (SEQ ID NO: 499) (SEQ ID NO: 504) (SEQ ID NO: 505)NO: 502) NO: 503) NO: 502) VH SDSAWFTY ARSDSAWFTY SDSAWFTY DSAWFTARSDSAWFT SDSAWFTY CDR3 (SEQ ID (SEQ ID (SEQ ID NO: 519) (SEQ ID NO:521) (SEQ ID NO: 522) (SEQ ID NO: 519) NO: 519) NO: 520) VL CDR VLSASSSVSYMY SSVSY SASSSVSYMY SSSVSY SYMYWY SASSSVSYMY Seq. CDR1 (SEQ ID(SEQ ID (SEQ ID NO: 535) (SEQ ID NO: 537) (SEQ ID NO: 538) (SEQ ID NO:535) NO: 535) NO: 536) VL DTSNLAS DTS DTSNLAS DTS LLIYDTSNLA DTSNLASCDR2 (SEQ ID (SEQ ID (SEQ ID NO: 550) (SEQ ID NO: 551) (SEQ ID NO: 552)(SEQ ID NO: 550) NO: 550) NO: 551) VL QQWNSYPPT QQWNSYPPT QQWNSYPPTWNSYPP QQWNSYPP QQWNSYPPT CDR3 (SEQ ID (SEQ ID (SEQ ID NO: 561) (SEQ IDNO: 562) (SEQ ID NO: 563) (SEQ ID NO: 561) NO: 561) NO: 561) VHSequence:EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQTHGKSLEWIGDINPNNGGPIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARSDSAWFTYWGQGTLVTVSA (SEQ ID NO: 482) VL Sequence:QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTFYSITISRMEAEDAATYYCQQWNSYPPTFGGGTKLEIK (SEQ ID NO: 483)

TABLE 23 Antibody P1H8 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH GYTFTDYYMN GYTFTDYY DYYMN GYTFTDY TDYYMNGYTFTDYYMN Seq. CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 490) (SEQ ID NO: 49)(SEQ ID NO: 491) (SEQ ID NO: 489) NO: 489) NO: 57) VH DINPNNGGTTYNINPNNGGT DINPNNGGTTYN PNNG WIGDINPNNGGT DINPNNGGTT CDR2 QKFKG (SEQ ID(SEQ ID QKFKG (SEQ ID (SEQ ID NO: 499) T (SEQ ID NO: (SEQ ID NO: 509)NO: 506) NO: 507) NO: 506) 508) VH QGPWYFDV ARQGPWYFDV QGPWYFDV GPWYFDARQGPWYFD QGPWYFDV CDR3 (SEQ ID (SEQ ID (SEQ ID NO: 523) (SEQ ID NO:525) (SEQ ID NO: 526) (SEQ ID NO: 523) NO: 523) NO: 524) VL CDR VLRSSQTIVHSNGY QTIVHSNGYTY RSSQTIVHSNGY SQTIVHSNGYTY VHSNGYTYLEWYRSSQTIVHSNGY Seq. CDR1 TYLE (SEQ ID (SEQ ID TYLE (SEQ ID (SEQ ID NO:541) (SEQ ID NO: 542) TYLE (SEQ ID NO: 539) NO: 540) NO: 539) NO: 539)VL KVSNRFS KVS KVSNRFS KVS LLIYKVSNRF KVSNRFS CDR2 (SEQ ID (SEQ ID (SEQID NO: 553) (SEQ ID NO: 554) (SEQ ID NO: 555) (SEQ ID NO: 553) NO: 553)NO: 554) VL FQGSHVPWT FQGSHVPWT FQGSHVPWT QGSHVPW FQGSHVPW FQGSHVPWTCDR3 (SEQ ID (SEQ ID (SEQ ID NO: 564) (SEQ ID NO: 565) (SEQ ID NO: 566)(SEQ ID NO: 564) NO: 564) NO: 564) VH Sequence:EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARQGPWYFDVWGTGTTVTVSS (SEQ ID NO: 484) VL Sequence:DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGYTYLEWYLQKPGQSPKWYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 485)

TABLE 24 Antibody P8G4 CDR Sequences Exemplary IMGT Kabat ChothiaContact AbM VH CDR VH GFSLTSYGVH GFSLTSYG SYGVH GFSLTSY TSYGVHGFSLTSYGVH Seq. CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 494) (SEQ ID NO: 495)(SEQ ID NO: 496) (SEQ ID NO: 492) NO: 492) NO: 493) VH VLWSGGSTDYNLWSGGST VLWSGGSTDYN GGS WLGVLWSGGST VLWSGGSTD CDR2 AAFIS (SEQ ID (SEQ IDAAFIS (SEQ ID (SEQ ID NO: 512) D (SEQ ID NO: (SEQ ID NO: 514) NO: 510)NO: 511) NO: 510) 513) VH NFGDY ARNFGDY NFGDY FGD ARNFGD NFGDY CDR3 (SEQID (SEQ ID (SEQ ID NO: 527) (SEQ ID NO: 529) (SEQ ID NO: 530) (SEQ IDNO: 527) NO: 527) NO: 528) VL CDR VL SASSRVSYMH SRVSY SASSRVSYMH SSRVSYSYMHWY SASSRVSYMH Seq. CDR1 (SEQ ID (SEQ ID (SEQ ID NO: 543) (SEQ ID NO:545) (SEQ ID NO: 546) (SEQ ID NO: 543) NO: 543) NO: 544) VL DTSKLAS DTSDTSKLAS DTS RWIYDTSKLA DTSKLAS CDR2 (SEQ ID (SEQ ID (SEQ ID NO: 556)(SEQ ID NO: 551) (SEQ ID NO: 557) (SEQ ID NO: 556) NO: 556) NO: 551) VLQQWNNNPPT QQWNNNPPT QQWNNNPPT WNNNPP QQWNNNPP QQWNNNPPT CDR3 (SEQ ID(SEQ ID (SEQ ID NO: 567) (SEQ ID NO: 568) (SEQ ID NO: 569) (SEQ ID NO:567) NO: 567) NO: 567) VH Sequence:QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLDWLGVLWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARNFGDYWGQGTSVTVSS (SEQ ID NO: 486) VL Sequence:QIVLTQSPAIMSASPGEKVTMTCSASSRVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNNNPPTFGAGTTLELK (SEQ ID NO: 487)

In some embodiments, the antibodies provided herein comprise a VH regionor VH domain In other embodiments, the antibodies provided hereincomprise a VL region or VL chain. In some embodiments, the antibodiesprovided herein have a combination of (i) a VH domain or VH region;and/or (ii) a VL domain or VL region.

In some embodiments, an antibody provided herein comprises or consistsof six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and/or VL CDR3 identified in Tables 1-24. In some embodiments, anantibody provided herein can comprise less than six CDRs. In someembodiments, the antibody comprises or consists of one, two, three,four, or five CDRs selected from the group consisting of VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables1-24. In some embodiments, the antibody comprises or consists of one,two, three, four, or five CDRs selected from the group consisting of VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of the murinemonoclonal antibody selected from the group consisting of: (a) theantibody designated 1C1; (b) the antibody designated 3P10; (c) theantibody designated 12A3; (d) the antibody designated 5F12; (e) theantibody designated 5A20; (f) the antibody designated 8D8; (g) theantibody designated 17J16; (h) t the antibody designated 25M22; (i) theantibody designated 2B8; (j) the antibody designated 22N5; (k) theantibody designated 2I23; (l) the antibody designated 6N16; (m) theantibody designated 1B3; (n) the antibody designated 19K19; (o) theantibody designated 2B3; (p) the antibody designated 8C10; (q) theantibody designated 2A9; (r) the antibody designated 24G2; (s) theantibody designated 6G9; (t) the antibody designated 2B11; (u) theantibody designated 1A3; (v) the antibody designated P1B6; (w) theantibody designated P1H8; or (x) the antibody designated P8G4 describedherein. Accordingly, in some embodiments, the antibody comprises orconsists of one, two, three four or five CDRs of anyone of the VH CDR1,VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables1-24.

In some embodiments, the antibodies provided herein comprise or consistof one or more (e.g., one, two or three) VH CDRs listed in Tables 1-24.In other embodiments, the antibodies provided herein comprise one ormore (e.g., one, two or three) VL CDRs listed in Tables 1-24. In yetother embodiments, the antibodies provided herein comprise one or more(e.g., one, two or three) VH CDRs listed in Tables 1-24 and one or moreVL CDRs listed in Tables 1-24. Accordingly, in certain embodiments, theantibodies comprise a VH CDR1 having the amino acid sequence of any oneof SEQ ID NOS: 57, 49, 57, 49, 221-296, 515-530, 488-493, 1795, 1796,488-493, 1795, 1796. In another embodiment, the antibodies comprise a VHCDR2 having the amino acid sequence of any one of SEQ ID NOS: 132-220,497-514. In another embodiment, the antibodies comprise a VH CDR3 havingthe amino acid sequence of any one of SEQ ID NOS: 57, 49, 57, 49,221-296, 515-530, 488-493, 1795, 1796, 488-493, 1795, 1796. In certainembodiments, the antibodies comprise a VH CDR1 and/or a VH CDR2 and/or aVH CDR3 independently selected from a VH CDR1, VH CDR2, VH CDR3 asdepicted in any one of the amino acid sequences depicted in Table 1-24.In certain embodiments, the antibodies comprise a VL CDR1 having theamino acid sequence of any one of SEQ ID NOS: 297-372, 531-546. Inanother embodiment, the antibodies comprise a VL CDR2 having the aminoacid sequence of any one of SEQ ID NOS: 373-422. In another embodiment,the antibodies comprise a VL CDR3 having the amino acid sequence of anyone of SEQ ID NOS: 423-479, 558-569. In certain embodiments, theantibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3independently selected from a VL CDR1, VL CDR2, VL CDR3 as depicted inany one of the amino acid sequences depicted in Tables 1-24.

Also provided herein are antibodies comprising one or more (e.g., one,two or three) VH CDRs and one or more (e.g., one, two or three) VL CDRslisted in Tables 1-24. In particular, provided herein is an antibodycomprising: a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493,1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR1 (SEQID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2(SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530,488-493, 1795, 1796) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VHCDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR1 (SEQ ID NOS: 297-372,531-546); a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR2 (SEQ IDNOS: 373-422); a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR3(SEQ ID NOS: 423-479, 558-569); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296,515-530, 488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372,531-546); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493,1795, 1796) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR3 (SEQ ID NOS:57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR3 (SEQ IDNOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530,488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VLCDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR1 (SEQ ID NOS: 57, 49,221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220,497-514) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57,49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS:132-220, 497-514) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569);a VH CDR2(SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296,515-530, 488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372,531-546), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2 (SEQID NOS: 373-422); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3(SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VLCDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49,221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372,531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57,49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS:297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VHCDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VLCDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569);a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS:297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR2 (SEQ IDNOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and aVL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220,497-514), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS:423-479, 558-569); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530,488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VLCDR2 (SEQ ID NOS: 373-422); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296,515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546)and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR3 (SEQ ID NOS: 57,49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS:373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ IDNOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530,488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VHCDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VHCDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49,221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2 (SEQ ID NOS:373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493,1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR3 (SEQID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296,515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514),a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS:373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493,1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ IDNOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); aVH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), aVH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR2 (SEQ ID NOS: 373-422)and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57,49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR3 (SEQ ID NOS: 57,49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS:297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR3 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ IDNOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); aVH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), aVH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), aVL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479,558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ IDNOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR2(SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296,515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546)and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS:132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530,488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296,515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514),a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), aVL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS:373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493,1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ IDNOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ IDNOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); aVH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), aVH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49,221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422)and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57,49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS:132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546), a VL CDR2(SEQ ID NOS: 373-422), and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); aVH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), aVH CDR3(SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VLCDR1 (SEQ ID NOS: 297-372, 531-546), a VL CDR2 (SEQ ID NOS: 373-422),and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS:132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530,488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546), a VLCDR2 (SEQ ID NOS: 373-422), and a VL CDR3 (SEQ ID NOS: 423-479,558-569); or any combination thereof of the VH CDRs (SEQ ID NOS: 41-296)and VL CDRs (SEQ ID NOS: 297-477) listed in Tables 1-24.

In certain embodiments, an antibody or fragment thereof described hereincomprises a humanized framework region (FR) sequence. In certainembodiments, an antibody or fragment thereof described herein comprisesa VH region comprising a VH FR1, a VH FR2, a VH FR3 and a VH FR4 aminoacid sequence depicted in Table 25; and/or (b) a VL region comprising aVL FR1, a VL FR2, a VL FR3 and a VL FR4 amino acid sequence depicted inTable 25.

TABLE 25 Exemplary Framework Sequences for Humanized Anti-GFRALAntibodies VH Clones Humanized SEQ ID NO: VH Framework 1 (FR1)QVQLQESGPGLVKPSETLSLTCTVS 1C1 570 QMQLQESGPGLVKPSETLSLTCTVS 1C1 571QVQLVQSGAEVKKPGSSVKVSCKAS 3P10, 5F12, 25M22, 572 17J16QIQLVQSGAEVKKPGSSVKVSCKAS 3P10 573 QVQLVQSGAEVKKPGATVKISCKVS 3P10 574QIQLVQSGAEVKKPGATVKISCKVS 3P10 575 QVQLVQSGAEVVKPGSSVKVSCKAS 5F12 576QVQLVQSGAEVKKPGASVKVSCKAS 5F12 577 EVQLVQSGAEVKKPGESLKISCKGS 25M22 578VH Framework 2 (FR2) WIRQPPGKGLEWIG 1C1 579 WIRQPPGKGLEWLG 1C1 580WVRQAPGQGLEWMG 3P10, 5F12, 25M22, 581 17J16 WVRQAPGKGLEWMG 3P10 582WVRQAPGQALEWMG 3P10 583 WVRQAPGQGLKWMG 3P10 584 WVRQAPGKALEWMG 3P10 585WVRQAPGKGLKWMG 3P10 586 WVRQAPGQALKWMG 3P10 587 WVRQAPGKALKWMG 3P10 588WVKQAPGQGLEWIG 5F12, 17J16 589 WVRQAPGQGLEWIG 5F12, 25M22, 17J16 590WVRQAPGQGLEWIA 5F12 591 WVKQAPGQGLEWIG 5F12, 17J16 592 WVKQAPGQGLEWIA5F12 593 WVRQAPGQGLEWIA 5F12 594 WVKQAPGQGLEWIA 5F12 595 WMQQAPGKGLEWIG3P10 596 WVRQAPGQRLEWMG 5F12 597 WVRQAPGQRLEWIG 5F12 598 WVRQAPGQRLEWMA5F12 599 WVRQAPGQRLEWIA 5F12 600 WVRQMPGKGLEWMG 25M22 601 WVRQMPGKGLEWIG25M22 602 VH Framework 3 (FR3) RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 603RLTISVDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 604RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 605RVTISVDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 606RVTISVDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 607RVTISVDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 608RVTISVDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 609RVTISVDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 610RVTISVDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 611RVTISVDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 612RVTISVDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 613RLTISKDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 614RLTISVDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 615RLTISVDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 616RLTISVDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 617RLTISVDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 618RLTISVDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 619RLTISVDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 620RLTISVDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 621RLTISVDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 622RVTISKDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 623RVTISKDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 624RVTISKDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 625RVTISKDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 626RVTISKDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 627RVTISKDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 628RVTISKDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 629RVTISKDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 630RVTISVDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 631RVTISVDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 632RVTISVDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 633RVTISVDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 634RVTISVDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 635RVTISVDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 636RVTISVDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 637RVTISVDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 638RVTISVDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 639RVTISVDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 640RVTISVDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 641RVTISVDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 642RVTISVDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 643RVTISVDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 644RVTISVDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 645RVTISVDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 646RVTISVDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 647RVTISVDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 648RVTISVDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 649RVTISVDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 650RVTISVDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 651RVTISVDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 652RVTISVDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 653RVTISVDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 654RVTISVDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 655RVTISVDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 656RVTISVDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 657RVTISVDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 658RLTISKDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 659RLTISKDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 660RLTISKDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 661RLTISKDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 662RLTISKDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 663RLTISKDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 664RLTISKDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 665RLTISKDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 666RLTISVDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 667RLTISVDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 668RLTISVDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 669RLTISVDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 670RLTISVDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 671RLTISVDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 672RLTISVDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 673RLTISVDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 674RLTISVDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 675RLTISVDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 676RLTISVDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 677RLTISVDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 678RLTISVDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 679RLTISVDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 680RLTISVDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 681RLTISVDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 682RLTISVDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 683RLTISVDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 684RLTISVDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 685RLTISVDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 686RLTISVDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 687RLTISVDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 688RLTISVDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 689RLTISVDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 690RLTISVDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 691RLTISVDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 692RLTISVDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 693RLTISVDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 694RVTISKDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 695RVTISKDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 696RVTISKDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 697RVTISKDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 698RVTISKDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 699RVTISKDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 700RVTISKDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 701RVTISKDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 702RVTISKDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 703RVTISKDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 704RVTISKDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 705RVTISKDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 706RVTISKDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 707RVTISKDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 708RVTISKDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 709RVTISKDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 710RVTISKDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 711RVTISKDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 712RVTISKDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 713RVTISKDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 714RVTISKDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 715RVTISKDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 716RVTISKDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 717RVTISKDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 718RVTISKDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 719RVTISKDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 720RVTISKDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 721RVTISKDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 722RVTISVDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 723RVTISVDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 724RVTISVDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 725RVTISVDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 726RVTISVDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 727RVTISVDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 728RVTISVDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 729RVTISVDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 730RVTISVDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 731RVTISVDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 732RVTISVDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 733RVTISVDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 734RVTISVDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 735RVTISVDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 736RVTISVDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 737RVTISVDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 738RVTISVDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 739RVTISVDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 740RVTISVDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 741RVTISVDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 742RVTISVDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 743RVTISVDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 744RVTISVDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 745RVTISVDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 746RVTISVDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 747RVTISVDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 748RVTISVDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 749RVTISVDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 750RVTISVDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 751RVTISVDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 752RVTISVDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 753RVTISVDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 754RVTISVDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 755RVTISVDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 756RVTISVDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 757RVTISVDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 758RVTISVDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 759RVTISVDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 760RVTISVDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 761RVTISVDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 762RVTISVDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 763RVTISVDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 764RVTISVDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 765RVTISVDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 766RVTISVDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 767RVTISVDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 768RVTISVDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 769RVTISVDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 770RVTISVDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 771RVTISVDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 772RVTISVDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 773RVTISVDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 774RVTISVDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 775RVTISVDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 776RVTISVDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 777RVTISVDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 778RLTISKDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 779RLTISKDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 780RLTISKDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 781RLTISKDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 782RLTISKDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 783RLTISKDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 784RLTISKDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 785RLTISKDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 786RLTISKDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 787RLTISKDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 788RLTISKDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 789RLTISKDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 790RLTISKDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 791RLTISKDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 792RLTISKDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 793RLTISKDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 794RLTISKDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 795RLTISKDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 796RLTISKDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 797RLTISKDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 798RLTISKDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 799RLTISKDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 800RLTISKDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 801RLTISKDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 802RLTISKDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 803RLTISKDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 804RLTISKDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 805RLTISKDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 806RLTISVDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 807RLTISVDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 808RLTISVDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 809RLTISVDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 810RLTISVDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 811RLTISVDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 812RLTISVDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 813RLTISVDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 814RLTISVDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 815RLTISVDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 816RLTISVDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 817RLTISVDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 818RLTISVDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 819RLTISVDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 820RLTISVDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 821RLTISVDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 822RLTISVDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 823RLTISVDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 824RLTISVDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 825RLTISVDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 826RLTISVDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 827RLTISVDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 828RLTISVDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 829RLTISVDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 830RLTISVDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 831RLTISVDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 832RLTISVDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 833RLTISVDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 834RLTISVDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 835RLTISVDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 836RLTISVDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 837RLTISVDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 838RLTISVDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 839RLTISVDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 840RLTISVDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 841RLTISVDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 842RLTISVDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 843RLTISVDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 844RLTISVDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 845RLTISVDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 846RLTISVDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 847RLTISVDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 848RLTISVDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 849RLTISVDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 850RLTISVDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 851RLTISVDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 852RLTISVDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 853RLTISVDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 854RLTISVDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 855RLTISVDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 856RLTISVDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 857RLTISVDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 858RLTISVDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 859RLTISVDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 860RLTISVDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 861RLTISVDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 862RVTISKDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 863RVTISKDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 864RVTISKDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 865RVTISKDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 866RVTISKDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 867RVTISKDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 868RVTISKDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 869RVTISKDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 870RVTISKDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 871RVTISKDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 872RVTISKDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 873RVTISKDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 874RVTISKDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 875RVTISKDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 876RVTISKDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 877RVTISKDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 878RVTISKDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 879RVTISKDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 880RVTISKDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 881RVTISKDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 882RVTISKDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 883RVTISKDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 884RVTISKDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 885RVTISKDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 886RVTISKDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 887RVTISKDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 888RVTISKDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 889RVTISKDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 890RVTISKDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 891RVTISKDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 892RVTISKDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 893RVTISKDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 894RVTISKDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 895RVTISKDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 896RVTISKDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 897RVTISKDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 898RVTISKDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 899RVTISKDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 900RVTISKDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 901RVTISKDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 902RVTISKDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 903RVTISKDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 904RVTISKDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 905RVTISKDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 906RVTISKDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 907RVTISKDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 908RVTISKDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 909RVTISKDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 910RVTISKDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 911RVTISKDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 912RVTISKDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 913RVTISKDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 914RVTISKDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 915RVTISKDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 916RVTISKDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 917RVTISKDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 918RVTISVDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 919RVTISVDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 920RVTISVDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 921RVTISVDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 922RVTISVDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 923RVTISVDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 924RVTISVDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 925RVTISVDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 926RVTISVDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 927RVTISVDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 928RVTISVDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 929RVTISVDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 930RVTISVDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 931RVTISVDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 932RVTISVDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 933RVTISVDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 934RVTISVDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 935RVTISVDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 936RVTISVDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 937RVTISVDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 938RVTISVDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 939RVTISVDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 940RVTISVDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 941RVTISVDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 942RVTISVDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 943RVTISVDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 944RVTISVDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 945RVTISVDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 946RVTISVDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 947RVTISVDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 948RVTISVDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 949RVTISVDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 950RVTISVDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 951RVTISVDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 952RVTISVDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 953RVTISVDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 954RVTISVDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 955RVTISVDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 956RVTISVDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 957RVTISVDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 958RVTISVDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 959RVTISVDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 960RVTISVDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 961RVTISVDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 962RVTISVDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 963RVTISVDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 964RVTISVDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 965RVTISVDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 966RVTISVDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 967RVTISVDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 968RVTISVDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 969RVTISVDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 970RVTISVDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 971RVTISVDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 972RVTISVDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 973RVTISVDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 974RVTISVDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 975RVTISVDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 976RVTISVDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 977RVTISVDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 978RVTISVDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 979RVTISVDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 980RVTISVDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 981RVTISVDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 982RVTISVDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 983RVTISVDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 984RVTISVDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 985RVTISVDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 986RVTISVDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 987RVTISVDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 988RLTISKDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 989RLTISKDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 990RLTISKDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 991RLTISKDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 992RLTISKDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 993RLTISKDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 994RLTISKDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 995RLTISKDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 996RLTISKDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 997RLTISKDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 998RLTISKDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 999RLTISKDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 1000RLTISKDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 1001RLTISKDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 1002RLTISKDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 1003RLTISKDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 1004RLTISKDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 1005RLTISKDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 1006RLTISKDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 1007RLTISKDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 1008RLTISKDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 1009RLTISKDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 1010RLTISKDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 1011RLTISKDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 1012RLTISKDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 1013RLTISKDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1014RLTISKDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 1015RLTISKDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 1016RLTISKDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 1017RLTISKDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1018RLTISKDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 1019RLTISKDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 1020RLTISKDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1021RLTISKDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 1022RLTISKDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1023RLTISKDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1024RLTISKDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 1025RLTISKDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 1026RLTISKDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 1027RLTISKDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1028RLTISKDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 1029RLTISKDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 1030RLTISKDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1031RLTISKDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 1032RLTISKDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1033RLTISKDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1034RLTISKDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 1035RLTISKDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 1036RLTISKDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1037RLTISKDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 1038RLTISKDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1039RLTISKDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1040RLTISKDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 1041RLTISKDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1042RLTISKDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1043RLTISKDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1044RLTISVDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 1045RLTISVDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 1046RLTISVDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 1047RLTISVDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 1048RLTISVDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1049RLTISVDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 1050RLTISVDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 1051RLTISVDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 1052RLTISVDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1053RLTISVDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 1054RLTISVDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 1055RLTISVDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1056RLTISVDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 1057RLTISVDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1058RLTISVDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1059RLTISVDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 1060RLTISVDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 1061RLTISVDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 1062RLTISVDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1063RLTISVDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 1064RLTISVDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 1065RLTISVDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1066RLTISVDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 1067RLTISVDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1068RLTISVDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1069RLTISVDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 1070RLTISVDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 1071RLTISVDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1072RLTISVDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 1073RLTISVDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1074RLTISVDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1075RLTISVDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 1076RLTISVDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1077RLTISVDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1078RLTISVDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1079RLTISVDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 1080RLTISVDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 1081RLTISVDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 1082RLTISVDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1083RLTISVDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 1084RLTISVDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 1085RLTISVDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1086RLTISVDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 1087RLTISVDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1088RLTISVDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1089RLTISVDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 1090RLTISVDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 1091RLTISVDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1092RLTISVDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 1093RLTISVDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1094RLTISVDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1095RLTISVDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 1096RLTISVDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1097RLTISVDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1098RLTISVDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1099RLTISVDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 1100RLTISVDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 1101RLTISVDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1102RLTISVDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 1103RLTISVDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1104RLTISVDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1105RLTISVDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 1106RLTISVDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1107RLTISVDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1108RLTISVDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1109RLTISVDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 1110RLTISVDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1111RLTISVDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1112RLTISVDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1113RLTISVDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1114RVTISKDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 1115RVTISKDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 1116RVTISKDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 1117RVTISKDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 1118RVTISKDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1119RVTISKDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 1120RVTISKDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 1121RVTISKDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 1122RVTISKDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1123RVTISKDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 1124RVTISKDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 1125RVTISKDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1126RVTISKDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 1127RVTISKDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1128RVTISKDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1129RVTISKDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 1130RVTISKDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 1131RVTISKDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 1132RVTISKDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1133RVTISKDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 1134RVTISKDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 1135RVTISKDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1136RVTISKDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 1137RVTISKDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1138RVTISKDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1139RVTISKDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 1140RVTISKDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 1141RVTISKDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1142RVTISKDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 1143RVTISKDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1144RVTISKDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1145RVTISKDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 1146RVTISKDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1147RVTISKDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1148RVTISKDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1149RVTISKDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 1150RVTISKDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 1151RVTISKDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 1152RVTISKDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1153RVTISKDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 1154RVTISKDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 1155RVTISKDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1156RVTISKDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 1157RVTISKDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1158RVTISKDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1159RVTISKDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 1160RVTISKDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 1161RVTISKDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1162RVTISKDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 1163RVTISKDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1164RVTISKDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1165RVTISKDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 1166RVTISKDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1167RVTISKDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1168RVTISKDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1169RVTISKDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 1170RVTISKDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 1171RVTISKDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1172RVTISKDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 1173RVTISKDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1174RVTISKDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1175RVTISKDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 1176RVTISKDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1177RVTISKDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1178RVTISKDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1179RVTISKDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 1180RVTISKDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1181RVTISKDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1182RVTISKDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1183RVTISKDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1184RVTISVDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1185RVTISVDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1186RVTISVDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1187RVTISVDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1188RVTISVDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1189RVTISVDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1190RVTISVDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1191RVTISVDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1192RVTISVDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1193RVTISVDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1194RVTISVDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1195RVTISVDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1196RVTISVDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1197RVTISVDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1198RVTISVDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1199RVTISVDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1200RVTISVDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1201RVTISVDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1202RVTISVDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1203RVTISVDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1204RVTISVDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1205RVTISVDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1206RVTISVDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1207RVTISVDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1208RVTISVDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1209RVTISVDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1210RVTISVDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1211RVTISVDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1212RVTISVDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1213RVTISVDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1214RVTISVDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1215RVTISVDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1216RVTISVDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1217RVTISVDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1218RVTISVDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1219RVTISVDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1220RVTISVDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1221RVTISVDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1222RVTISVDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1223RVTISVDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1224RVTISVDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1225RVTISVDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1226RVTISVDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1227RVTISVDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1228RVTISVDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1229RVTISVDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1230RVTISVDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1231RVTISVDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1232RVTISVDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1233RVTISVDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1234RVTISVDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1235RVTISVDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1236RVTISVDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1237RVTISVDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1238RVTISVDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1239RVTISVDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1240RLTISKDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 1241RLTISKDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 1242RLTISKDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 1243RLTISKDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 1244RLTISKDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1245RLTISKDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 1246RLTISKDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 1247RLTISKDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 1248RLTISKDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1249RLTISKDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 1250RLTISKDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 1251RLTISKDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1252RLTISKDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 1253RLTISKDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1254RLTISKDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1255RLTISKDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 1256RLTISKDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 1257RLTISKDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 1258RLTISKDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1259RLTISKDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 1260RLTISKDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 1261RLTISKDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1262RLTISKDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 1263RLTISKDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1264RLTISKDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1265RLTISKDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 1266RLTISKDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 1267RLTISKDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1268RLTISKDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 1269RLTISKDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1270RLTISKDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1271RLTISKDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 1272RLTISKDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1273RLTISKDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1274RLTISKDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1275RLTISKDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 1276RLTISKDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 1277RLTISKDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 1278RLTISKDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1279RLTISKDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 1280RLTISKDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 1281RLTISKDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1282RLTISKDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 1283RLTISKDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1284RLTISKDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1285RLTISKDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 1286RLTISKDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 1287RLTISKDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1288RLTISKDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 1289RLTISKDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1290RLTISKDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1291RLTISKDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 1292RLTISKDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1293RLTISKDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1294RLTISKDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1295RLTISKDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 1296RLTISKDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 1297RLTISKDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1298RLTISKDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 1299RLTISKDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1300RLTISKDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1301RLTISKDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 1302RLTISKDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1303RLTISKDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1304RLTISKDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1305RLTISKDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 1306RLTISKDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1307RLTISKDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1308RLTISKDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1309RLTISKDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1310RLTISVDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1311RLTISVDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1312RLTISVDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1313RLTISVDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1314RLTISVDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1315RLTISVDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1316RLTISVDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1317RLTISVDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1318RLTISVDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1319RLTISVDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1320RLTISVDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1321RLTISVDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1322RLTISVDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1323RLTISVDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1324RLTISVDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1325RLTISVDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1326RLTISVDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1327RLTISVDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1328RLTISVDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1329RLTISVDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1330RLTISVDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1331RLTISVDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1332RLTISVDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1333RLTISVDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1334RLTISVDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1335RLTISVDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1336RLTISVDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1337RLTISVDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1338RLTISVDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1339RLTISVDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1340RLTISVDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1341RLTISVDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1342RLTISVDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1343RLTISVDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1344RLTISVDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1345RLTISVDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1346RLTISVDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1347RLTISVDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1348RLTISVDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1349RLTISVDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1350RLTISVDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1351RLTISVDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1352RLTISVDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1353RLTISVDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1354RLTISVDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1355RLTISVDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1356RLTISVDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1357RLTISVDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1358RLTISVDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1359RLTISVDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1360RLTISVDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1361RLTISVDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1362RLTISVDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1363RLTISVDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1364RLTISVDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1365RLTISVDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1366RVTISKDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1367RVTISKDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1368RVTISKDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1369RVTISKDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1370RVTISKDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1371RVTISKDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1372RVTISKDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1373RVTISKDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1374RVTISKDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1375RVTISKDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1376RVTISKDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1377RVTISKDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1378RVTISKDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1379RVTISKDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1380RVTISKDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1381RVTISKDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1382RVTISKDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1383RVTISKDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1384RVTISKDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1385RVTISKDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1386RVTISKDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1387RVTISKDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1388RVTISKDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1389RVTISKDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1390RVTISKDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1391RVTISKDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1392RVTISKDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1393RVTISKDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1394RVTISKDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1395RVTISKDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1396RVTISKDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1397RVTISKDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1398RVTISKDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1399RVTISKDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1400RVTISKDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1401RVTISKDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1402RVTISKDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1403RVTISKDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1404RVTISKDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1405RVTISKDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1406RVTISKDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1407RVTISKDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1408RVTISKDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1409RVTISKDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1410RVTISKDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1411RVTISKDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1412RVTISKDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1413RVTISKDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1414RVTISKDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1415RVTISKDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1416RVTISKDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1417RVTISKDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1418RVTISKDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1419RVTISKDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1420RVTISKDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1421RVTISKDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1422RVTISVDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1423RVTISVDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1424RVTISVDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1425RVTISVDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1426RVTISVDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1427RVTISVDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1428RVTISVDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1429RVTISVDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1430RVTISVDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1431RVTISVDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1432RVTISVDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1433RVTISVDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1434RVTISVDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1435RVTISVDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1436RVTISVDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1437RVTISVDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1438RVTISVDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1439RVTISVDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1440RVTISVDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1441RVTISVDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1442RVTISVDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1443RVTISVDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1444RVTISVDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1445RVTISVDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1446RVTISVDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1447RVTISVDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1448RVTISVDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1449RVTISVDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1450RLTISKDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1451RLTISKDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1452RLTISKDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1453RLTISKDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1454RLTISKDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1455RLTISKDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1456RLTISKDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1457RLTISKDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1458RLTISKDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1459RLTISKDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1460RLTISKDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1461RLTISKDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1462RLTISKDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1463RLTISKDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1464RLTISKDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1465RLTISKDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1466RLTISKDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1467RLTISKDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1468RLTISKDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1469RLTISKDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1470RLTISKDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1471RLTISKDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1472RLTISKDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1473RLTISKDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1474RLTISKDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1475RLTISKDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1476RLTISKDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1477RLTISKDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1478RLTISKDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1479RLTISKDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1480RLTISKDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1481RLTISKDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1482RLTISKDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1483RLTISKDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1484RLTISKDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1485RLTISKDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1486RLTISKDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1487RLTISKDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1488RLTISKDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1489RLTISKDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1490RLTISKDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1491RLTISKDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1492RLTISKDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1493RLTISKDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1494RLTISKDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1495RLTISKDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1496RLTISKDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1497RLTISKDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1498RLTISKDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1499RLTISKDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1500RLTISKDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1501RLTISKDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1502RLTISKDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1503RLTISKDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1504RLTISKDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1505RLTISKDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1506RLTISVDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1507RLTISVDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1508RLTISVDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1509RLTISVDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1510RLTISVDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1511RLTISVDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1512RLTISVDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1513RLTISVDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1514RLTISVDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1515RLTISVDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1516RLTISVDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1517RLTISVDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1518RLTISVDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1519RLTISVDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1520RLTISVDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1521RLTISVDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1522RLTISVDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1523RLTISVDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1524RLTISVDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1525RLTISVDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1526RLTISVDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1527RLTISVDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1528RLTISVDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1529RLTISVDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1530RLTISVDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1531RLTISVDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1532RLTISVDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1533RLTISVDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1534RVTISKDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1535RVTISKDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1536RVTISKDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1537RVTISKDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1538RVTISKDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1539RVTISKDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1540RVTISKDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1541RVTISKDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1542RVTISKDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1543RVTISKDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1544RVTISKDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1545RVTISKDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1546RVTISKDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1547RVTISKDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1548RVTISKDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1549RVTISKDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1550RVTISKDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1551RVTISKDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1552RVTISKDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1553RVTISKDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1554RVTISKDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1555RVTISKDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1556RVTISKDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1557RVTISKDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1558RVTISKDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1559RVTISKDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1560RVTISKDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1561RVTISKDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1562RVTISVDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1563RVTISVDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1564RVTISVDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1565RVTISVDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1566RVTISVDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1567RVTISVDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1568RVTISVDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1569RVTISVDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1570RLTISKDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1571RLTISKDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1572RLTISKDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1573RLTISKDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1574RLTISKDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1575RLTISKDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1576RLTISKDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1577RLTISKDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1578RLTISKDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1579RLTISKDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1580RLTISKDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1581RLTISKDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1582RLTISKDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1583RLTISKDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1584RLTISKDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1585RLTISKDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1586RLTISKDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1587RLTISKDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1588RLTISKDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1589RLTISKDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1590RLTISKDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1591RLTISKDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1592RLTISKDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1593RLTISKDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1594RLTISKDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1595RLTISKDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1596RLTISKDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1597RLTISKDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1598RLTISVDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1599RLTISVDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1600RLTISVDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1601RLTISVDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1602RLTISVDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1603RLTISVDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1604RLTISVDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1605RLTISVDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1606RVTISKDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1607RVTISKDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1608RVTISKDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1609RVTISKDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1610RVTISKDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1611RVTISKDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1612RVTISKDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1613RVTISKDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1614RVTISVDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1615RLTISKDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1616RLTISKDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1617RLTISKDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1618RLTISKDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1619RLTISKDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1620RLTISKDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1621RLTISKDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1622RLTISKDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1623RLTISVDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1624RVTISKDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1625RLTISKDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1626RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 1627RLTISRDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 1628RVTISRDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 1629RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 1630RVTISRDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 1631RLTISRDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 1632RLTISRDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 1633RLTISRDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 1634RVTISRDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 1635RVTISRDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 1636RVTISRDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 1637RLTISRDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 1638RLTISRDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 1639RLTISRDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 1640RVTISRDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 1641RLTISRDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 1642RVTITADESTSTAYMELSSLRSEDTAVYYCAR 3P10, 5F12, 25M22, 1643 17J16RFTITADESTSTAYMELSSLRSEDTAVYYCAR 3P10 1644RVTFTADESTSTAYMELSSLRSEDTAVYYCAR 3P10 1645RVTITLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1646RVTITADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1647RVTITADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1648RFTFTADESTSTAYMELSSLRSEDTAVYYCAR 3P10 1649RFTITLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1650RFTITADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1651RFTITADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1652RVTFTLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1653RVTFTADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1654RVTFTADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1655RVTITLDESTSTAYMELSNLRSEDTAVYYCAR 3P10 1656RVTITLDESTSTAYMELSSLRSEDTAVFYCAR 3P10 1657RVTITADESTSTAYMELSNLRSEDTAVFYCAR 3P10 1658RFTFTLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1659RFTFTADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1660RFTFTADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1661RVTFTLDESTSTAYMELSNLRSEDTAVYYCAR 3P10 1662RVTFTLDESTSTAYMELSSLRSEDTAVFYCAR 3P10 1663RVTFTADESTSTAYMELSNLRSEDTAVFYCAR 3P10 1664RVTITLDESTSTAYMELSNLRSEDTAVFYCAR 3P10 1665RFTFTLDESTSTAYMELSNLRSEDTAVYYCAR 3P10 1666RFTFTLDESTSTAYMELSSLRSEDTAVFYCAR 3P10 1667RFTFTADESTSTAYMELSNLRSEDTAVFYCAR 3P10 1668RVTFTLDESTSTAYMELSNLRSEDTAVFYCAR 3P10 1669RFTFTLDESTSTAYMELSNLRSEDTAVFYCAR 3P10 1670RVTLTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1671RFTLTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1672RVTFTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1673RVTLTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1674RFTFTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1675RFTLTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1676RVTFTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1677RFTFTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1678RATITADESTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22, 17J16 1679RVTLTADESTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22, 17J16 1680RVTITADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1681RATLTADESTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22, 17J16 1682RATITADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1683RVTLTADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1684RATLTADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1685RATITADESTSTAYMELSSLRSEDTAVYYCAR 17J16 1686RVTITADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1687RVTITADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1688RVTITADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1689RATITADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1690RATITADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1691RATITADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1692RVTLTADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1693RVTLTADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1694RVTLTADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1695RVTITADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1696RVTITADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1697RVTITADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1698RATLTADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1699RATLTADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1700RATLTADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1701RATITADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1702RATITADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1703RATITADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1704RVTLTADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1705RVTLTADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1706RVTLTADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1707RVTITADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1708RATLTADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1709RATLTADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1710RATLTADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1711RATITADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1712RVTLTADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1713RATLTADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1714RVTITRDTSASTAYMELSSLRSEDTAVYYCAR 5F12 1715RATITRDESASTAYMELSSLRSEDTAVYYCAR 5F12 1716RVTLTRDESASTAYMELSSLRSEDTAVYYCAR 5F12 1717RVTITRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1718RATLTRDESASTAYMELSSLRSEDTAVYYCAR 5F12 1719RATITRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1720RVTLTRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1721RATLTRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1722QVTISADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1723QATISADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1724QVTLSADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1725QATLSADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1726 VH Framework 4 (FR4)WGQGTLVTVSS 1C1, 3P10, 5F12 1727 WGQGTTVTVSS 3P10, 25M22, 17J16 1728 VLHumanized Clone SEQ ID NO: VL Framework 1 (FR1) DVVMTQSPLSLPVTLGQPASISC1C1, 3P10, 5F12, 1729 25M22, 17J16 DVVLTQSPLSLPVTLGQPASISC 1C1, 3P10,5F12, 1730 25M22, 17J16 DVVLTQSPLSLPVTLGDPASISC 1C1 1731DVVLTQSPLSLPVTLGDPASISC 1C1 1732 DIVMTQSPLSLPVTLGQPASISC 3P10, 5F12 1733DIVLTQSPLSLPVTLGQPASISC 3P10, 5F12 1734 DVAMTQSPLSLPVTLGQPASISC 17J161735 DVALTQSPLSLPVTLGQPASISC 17J16 1736 DVVLTQTPLSLPVSPGDQASISC 1C1 1737DIVMTQTPLSLPVSPGDQASISC 1C1 1738 DIVMTQTPLSLPVSPGDQASISC 1C1 1739DIVMTQTPLSLPVSPGDQASISC 1C1 1740 DIELTQSPASLAVSLGQRATISC 3P10 1741DIVLTQSPASLAVSLGQRATISC 3P10 1742 DIVMTQSPDSLAVSLGERATINC 5F12 1743DIVLTQSPDSLAVSLGERATINC 5F12 1744 DIQMTQSPSSLSASVGDRVTITC 5F12 1745DIQLTQSPSSLSASVGDRVTITC 5F12 1746 EIVLTQSPATLSLSPGERATLSC 5F12 1747EIVLTQSPATLSVSPGERATLSC 5F12 1748 EIVLTQSPGTLSLSPGERATLSC 25M22 1749EVVLTQSPGTLSLSPGERATLSC 25M22 1750 VL Framework 2 (FR2) WFQQRPGQSPRRLIY1C1, 3P10, 5F12, 1751 25M22, 17J16 WYQQRPGQSPRRLIY 1C1, 5F12, 25M22 1752WFQQKPGQSPRRLIY 1C1 1753 WFQQRPGQSPKRLIY 1C1, 17J16 1754 WFQQRPGQSPRLLIY1C1, 3P10, 5F12, 1755 25M22 WYQQKPGQSPRRLIY 1C1 1756 WYQQRPGQSPKRLIY 1C11757 WYQQRPGQSPRLLIY 1C1, 5F12, 25M22 1758 WFQQKPGQSPKRLIY 1C1 1759WFQQKPGQSPRLLIY 1C1 1760 WFQQRPGQSPKLLIY 1C1 1761 WYQQKPGQSPKRLIY 1C11762 WYQQKPGQSPRLLIY 1C1 1763 WYQQRPGQSPKLLIY 1C1 1764 WFQQKPGQSPKLLIY1C1 1765 WYQQKPGQSPKLLIY 1C1 1766 WLQQRPGQSPRRLIY 17J16 1767WLQQRPGQSPKRLIY 17J16 1768 WFQQRPGQSPRRLIF 25M22 1769 WYQQRPGQSPRRLIF25M22 1770 WFQQRPGQSPRLLIF 25M22 1771 WYQQRPGQSPRLLIF 25M22 1772WYLQKPGQSPKLLIY 1C1 1773 WYQQKPGQPPKLLIY 3P10, 5F12 1774 WYQQKPGKAPKLLIY5F12 1775 WYQQKPGQAPRLLIY 5F12, 25M22 1776 WYQQKPGQAPRLLIF 25M22 1777 VLFramework 3 (FR3) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 1C1, 3P10, 5F12, 177825M22, 17J16 GVPDRFSGSGSGADFTLKISRVEAEDVGVYYC 17J16 1779GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC 1C1, 3P10 1780GVPDRFSGSGSRTDFTLKISRVEAEDVGVYYC 5F12 1781GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 5F12 1782GVPDRFSGSGSRTDFTLTISSLQAEDVAVYYC 5F12 1783GVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC 5F12 1784GVPDRFSGSGSRTDFTLTISSVQAEDVAVYYC 5F12 1785GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 5F12 1786GVPSRFSGSGSGTDFTLTISSVQPEDFATYYC 5F12 1787GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 5F12 1788GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC 5F12 1789GIPARFSGSGSGTDFTLTISSVEPEDFAVYYC 5F12 1790GVPARFSGSGSGTDFTLTISSVEPEDFAVYYC 5F12 1791GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 25M22 1792 VL Framework 4 (FR4)FGGGTKVEIK 1C1, 3P10, 5F12, 1793 25M22, 17J16 FGSGTKLEIK 1C1, 3P10 1794

In certain embodiments, an antibody or fragment thereof described hereincomprises a VH region that comprises: (1) a VH FR1 having an amino acidsequence selected from SEQ ID NOS: 570-578; (2) a VH FR2 having an aminoacid sequence selected from SEQ ID NOS: 579-602; (3) a VH FR3 having anamino acid sequence selected from SEQ ID NOS: 603-1726; and/or (4) a VHFR4 having an amino acid selected from SEQ ID NOS: 1727-1728.Accordingly, in some aspects, the humanized antibody comprises a VHregion that includes a VH FR1 having an amino acid sequence selectedfrom SEQ ID NOS: 570-578. In some aspects, the humanized antibodycomprises a VH region that includes a VH FR2 having an amino acidsequence selected from SEQ ID NOS: 579-602. In some aspects, thehumanized antibody comprises a VH region that includes a VH FR3 havingan amino acid sequence selected from SEQ ID NOS: 603-1726. In someaspects, the humanized antibody comprises a VH region that includes a VHFR4 having an amino acid selected from SEQ ID NOS: 1727-1728.

In certain embodiments, an antibody or fragment thereof described hereincomprises a VL region that comprises: (1) a VL FR1 having an amino acidsequence selected from SEQ ID NOS: 1729-1750; (2) a VL FR2 having anamino acid sequence selected from SEQ ID NOS: 1751-1777; (3) a VL FR3having an amino acid sequence selected from SEQ ID NOS: 1778-1792;and/or (4) a VL FR4 having an amino acid selected from SEQ ID NOS:1793-1794. Accordingly, in some aspects, the humanized antibodycomprises a VL region that includes a VL FR1 having an amino acidsequence selected from SEQ ID NOS: 1729-1750. In some aspects, thehumanized antibody comprises a VL region that includes a VL FR2 havingan amino acid sequence selected from SEQ ID NOS: 1751-1777. In someaspects, the humanized antibody comprises a VL region that includes a VLFR3 having an amino acid sequence selected from SEQ ID NOS: 1778-1792.In some aspects, the humanized antibody comprises a VL region thatincludes a VL FR4 having an amino acid selected from SEQ ID NOS:1793-1794.

In certain embodiments, an antibody or fragment thereof described hereincomprises a VH region and a VL region, wherein the VH region furthercomprises: (1) a VH FR1 having an amino acid sequence selected from SEQID NOS: 570-578; (2) a VH FR2 having an amino acid sequence selectedfrom SEQ ID NOS: 579-602; (3) a VH FR3 having an amino acid sequenceselected from SEQ ID NOS: 603-1726; and/or (4) a VH FR4 having an aminoacid sequence of SEQ ID NOS: 1727-1728; and wherein the VL regionfurther comprises: (1) a VL FR1 having an amino acid sequence selectedfrom SEQ ID NOS: 1729-1750; (2) a VL FR2 having an amino acid sequenceselected from SEQ ID NOS: 1751-1777; (3) a VL FR3 having an amino acidsequence selected from SEQ ID NOS: 1778-1792; and/or (4) a VL FR4 havingan amino acid selected from SEQ ID NOS: 1793-1794.

Also provided herein are antibodies comprising one or more (e.g., one,two, three or four) VH FRs and one or more (e.g., one, two, three orfour) VL FRs listed in Table 25. In particular, provided herein is anantibody comprising: a VH FR1 (SEQ ID NOS:570-578) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR2 (SEQ ID NOS:579-602) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ IDNOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ IDNOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ IDNOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ IDNOS:1729-1750); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ IDNOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ IDNOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729- 1750), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ IDNOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ IDNOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ IDNOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ IDNOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ IDNOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ IDNOS:1727-1728), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ IDNOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ IDNOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ IDNOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ IDNOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ IDNOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ IDNOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ IDNOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ IDNOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ IDNOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); or any combinationthereof of the VH FRs (SEQ ID NOS: 478-1636) and VL FRs (SEQ ID NOS:1637-1702) listed in Tables 25.

In yet another aspect, antibodies are provided that compete with one ofthe exemplified antibodies or functional fragments for binding to GFRAL.Such antibodies may also bind to the same epitope as one of the hereinexemplified antibodies, or an overlapping epitope. Antibodies andfragments that compete with or bind to the same epitope as theexemplified antibodies are expected to show similar functionalproperties. The exemplified antigen binding proteins and fragmentsinclude those with the VH and VL regions, and CDRs provided herein,including those in Tables 1-24. Thus, as a specific example, theantibodies that are provided include those that compete with an antibodycomprising: (a) 1, 2, 3, 4, 5 or all 6 of the CDRs listed for anantibody listed in Tables 1-24; (b) a VH and a VL selected from the VHand a VL regions listed for an antibody listed in Tables 1-24; or (c)two light chains and two heavy chains comprising a VH and a VL asspecified for an antibody listed in Tables 1-24.

In certain embodiments, antibodies of the compositions and methods ofusing the antibodies provided herein include those anti-GFRAL monoclonalantibodies described herein, e.g., in the Examples section. Anyanti-GFRAL antibody can be used in any of the methods provided herein.In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:1, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VH region comprisingthe amino acid sequence of SEQ ID NO: 3, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:5, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VH region comprisingthe amino acid sequence of SEQ ID NO: 7, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:9, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VH region comprisingthe amino acid sequence of SEQ ID NO: 11, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VH region comprising the amino acid sequence of SEQID NO: 13, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 15, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VH region comprising the amino acidsequence of SEQ ID NO: 17, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:17, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VH region comprisingthe amino acid sequence of SEQ ID NO: 19, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VH region comprising the amino acid sequence of SEQID NO: 21, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 23, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VH region comprising the amino acidsequence of SEQ ID NO: 25, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:27, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VH region comprisingthe amino acid sequence of SEQ ID NO: 29, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VH region comprising the amino acid sequence of SEQID NO: 31, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 33, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VH region comprising the amino acidsequence of SEQ ID NO: 35, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:37, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VH region comprisingthe amino acid sequence of SEQ ID NO: 39, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VH region comprising the amino acid sequence of SEQID NO: 480, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 482, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VH region comprising the amino acidsequence of SEQ ID NO: 484, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:486, or a humanized variant thereof.

In some embodiments of the various methods provided herein, the antibodycomprises a VL region comprising the amino acid sequence of SEQ ID NO:2, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VL region comprisingthe amino acid sequence of SEQ ID NO: 4, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VL region comprising the amino acid sequence of SEQ ID NO:6, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VL region comprisingthe amino acid sequence of SEQ ID NO: 8, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VL region comprising the amino acid sequence of SEQ ID NO:10, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VL region comprisingthe amino acid sequence of SEQ ID NO: 12, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VL region comprising the amino acid sequence of SEQID NO: 14, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VL regioncomprising the amino acid sequence of SEQ ID NO: 16, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VL region comprising the amino acidsequence of SEQ ID NO: 18, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VL region comprising the amino acid sequence of SEQ ID NO:20, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VL region comprisingthe amino acid sequence of SEQ ID NO: 22, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VL region comprising the amino acid sequence of SEQID NO: 24, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VL regioncomprising the amino acid sequence of SEQ ID NO: 26, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VL region comprising the amino acidsequence of SEQ ID NO: 28, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VL region comprising the amino acid sequence of SEQ ID NO:30, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VL region comprisingthe amino acid sequence of SEQ ID NO: 32, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VL region comprising the amino acid sequence of SEQID NO: 34, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VL regioncomprising the amino acid sequence of SEQ ID NO: 36, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VL region comprising the amino acidsequence of SEQ ID NO: 38, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VL region comprising the amino acid sequence of SEQ ID NO:40, or a humanized variant thereof. In some embodiments of the variousmethods provided herein, the antibody comprises a VL region comprisingthe amino acid sequence of SEQ ID NO: 481, or a humanized variantthereof. In some embodiments of the various methods provided herein, theantibody comprises a VL region comprising the amino acid sequence of SEQID NO: 483, or a humanized variant thereof. In some embodiments of thevarious methods provided herein, the antibody comprises a VL regioncomprising the amino acid sequence of SEQ ID NO: 485, or a humanizedvariant thereof. In some embodiments of the various methods providedherein, the antibody comprises a VL region comprising the amino acidsequence of SEQ ID NO: 487, or a humanized variant thereof.

In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:1, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 2, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:3, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 4, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:5, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 6, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:7, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 8, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:9, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 10, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:11, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 12, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:13, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 14, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:15, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 16, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:17, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 18, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:19, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 20, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:21, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 22, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:23, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 24, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:25, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 26, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:27, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 28, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:29, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 30, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:31, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 32, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:33, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 34, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:35, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 36, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:37, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 38, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:39, or a humanized variant thereof, and a VL region comprising the aminoacid sequence of SEQ ID NO: 40, or a humanized variant thereof. In someembodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:480, or a humanized variant thereof, and a VL region comprising theamino acid sequence of SEQ ID NO: 481, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:482, or a humanized variant thereof, and a VL region comprising theamino acid sequence of SEQ ID NO: 483, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:484, or a humanized variant thereof, and a VL region comprising theamino acid sequence of SEQ ID NO: 485, or a humanized variant thereof.In some embodiments of the various methods provided herein, the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:486, or a humanized variant thereof, and a VL region comprising theamino acid sequence of SEQ ID NO: 487, or a humanized variant thereof.

In some embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 1 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 2. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 3 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 4. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 5 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 6. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 7 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 8. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 9 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 10. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 11 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 12. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 13 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 14. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 15 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 16. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 17 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 18. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 19 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 20. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 21 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 22. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 23 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 24. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 25 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 26. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 27 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 28. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 29 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 30. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 31 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 32. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 33 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 34. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 35 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 36. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 37 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 38. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 39 and a VL CDR1, VL CDR2, and VL CDR3of a VL region comprising the amino acid sequence of SEQ ID NO: 40. Insome embodiments of the various methods provided herein, the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having theamino acid sequence of SEQ ID NO: 480 and a VL CDR1, VL CDR2, and VLCDR3 of a VL region comprising the amino acid sequence of SEQ ID NO:481. In some embodiments of the various methods provided herein, theantibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain havingthe amino acid sequence of SEQ ID NO: 482 and a VL CDR1, VL CDR2, and VLCDR3 of a VL region comprising the amino acid sequence of SEQ ID NO:483. In some embodiments of the various methods provided herein, theantibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain havingthe amino acid sequence of SEQ ID NO: 484 and a VL CDR1, VL CDR2, and VLCDR3 of a VL region comprising the amino acid sequence of SEQ ID NO:485. In some embodiments of the various methods provided herein, theantibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain havingthe amino acid sequence of SEQ ID NO: 486 and a VL CDR1, VL CDR2, and VLCDR3 of a VL region comprising the amino acid sequence of SEQ ID NO:487. Other VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 sequences listed described herein are also contemplatedfor use in the various methods provided herein.

1. Polyclonal Antibodies

The antibodies of the present disclosure may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include a GFRALpolypeptide or a fusion protein thereof. It may be useful to conjugatethe immunizing agent to a protein known to be immunogenic in the mammalbeing immunized or to immunize the mammal with the protein and one ormore adjuvants. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Ribi, CpG, Poly 1C, Freund's completeadjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetictrehalose dicorynomycolate). The immunization protocol may be selectedby one skilled in the art without undue experimentation. The mammal canthen be bled, and the serum assayed for GFRAL antibody titer. Ifdesired, the mammal can be boosted until the antibody titer increases orplateaus. Additionally or alternatively, lymphocytes may be obtainedfrom the immunized animal for fusion and the preparation of monoclonalantibodies from hybridoma as described below.

2. Monoclonal Antibodies

The antibodies of the present disclosure may alternatively be monoclonalantibodies. Monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp.59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as SP-2 and derivatives, for example,X63-Ag8-653 cells available from the American Type Culture Collection,Manassas, Va., USA and those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J., Immunol., 133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., N.Y., 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA). The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis described in Munson etal., Anal. Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp.59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal, for example, by i.p.injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In some embodiments, an antibody that binds a GFRAL epitope comprises anamino acid sequence of a VH domain and/or an amino acid sequence of a VLdomain encoded by a nucleotide sequence that hybridizes to (1) thecomplement of a nucleotide sequence encoding any one of the VH and/or VLdomain described herein under stringent conditions (e.g., hybridizationto filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about50-65° C.) under highly stringent conditions (e.g., hybridization tofilter-bound nucleic acid in 6×SSC at about 45° C. followed by one ormore washes in 0.1×SSC/0.2% SDS at about 68° C.), or under otherstringent hybridization conditions which are known to those of skill inthe art (see, for example, Ausubel, F. M. et al., eds., 1989, CurrentProtocols in Molecular Biology, Vol. I, Green Publishing Associates,Inc. and John Wiley & Sons, Inc., N.Y. at pages 6.3.1-6.3.6 and 2.10.3).

In some embodiments, an antibody that binds a GFRAL epitope comprises anamino acid sequence of a VH CDR or an amino acid sequence of a VL CDRencoded by a nucleotide sequence that hybridizes to the complement of anucleotide sequence encoding any one of the VH CDRs and/or VL CDRsdepicted in Tables 1-24 under stringent conditions (e.g., hybridizationto filter-bound DNA in 6× SSC at about 45° C. followed by one or morewashes in 0.2× SSC/0.1% SDS at about 50-65° C.), under highly stringentconditions (e.g., hybridization to filter-bound nucleic acid in 6× SSCat about 45° C. followed by one or more washes in 0.1× SSC/0.2% SDS atabout 68° C.), or under other stringent hybridization conditions whichare known to those of skill in the art (see, for example, Ausubel, F.M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc. and John Wiley & Sons, Inc., N.Y. atpages 6.3.1-6.3.6 and 2.10.3).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in, for example, Antibody Phage Display: Methods andProtocols, P. M. O'Brien and R. Aitken, eds, Humana Press, Totawa N.J.,2002. In principle, synthetic antibody clones are selected by screeningphage libraries containing phage that display various fragments ofantibody variable region (Fv) fused to phage coat protein. Such phagelibraries are screened for against the desired antigen. Clonesexpressing Fv fragments capable of binding to the desired antigen areadsorbed to the antigen and thus separated from the non-binding clonesin the library. The binding clones are then eluted from the antigen, andcan be further enriched by additional cycles of antigenadsorption/elution.

Variable domains can be displayed functionally on phage, either assingle-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed, for example, in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., supra. Libraries from immunized sources provide high-affinityantibodies to the immunogen without the requirement of constructinghybridomas. Alternatively, the naive repertoire can be cloned to providea single source of human antibodies to a wide range of non-self and alsoself antigens without any immunization as described by Griffiths et al.,EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be madesynthetically by cloning the unrearranged V-gene segments from stemcells, and using PCR primers containing random sequence to encode thehighly variable CDR3 regions and to accomplish rearrangement in vitro asdescribed, for example, by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, GFRAL, (e.g., a GFRAL polypeptide,fragment or epitope) can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other method for panning display libraries. Theselection of antibodies with slow dissociation kinetics (e.g., goodbinding affinities) can be promoted by use of long washes and monovalentphage display as described in Bass et al., Proteins, 8: 309-314 (1990)and in WO 92/09690, and a low coating density of antigen as described inMarks et al., Biotechnol., 10: 779-783 (1992).

Anti-GFRAL antibodies can be obtained by designing a suitable antigenscreening procedure to select for the phage clone of interest followedby construction of a full length anti-GFRAL antibody clone using VHand/or VL sequences (e.g., the Fv sequences), or various CDR sequencesfrom VH and VL sequences, from the phage clone of interest and suitableconstant region (e.g., Fc) sequences described in Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NIHPublication 91-3242, Bethesda Md. (1991), vols. 1-3.

3. Antibody Fragments

The present disclosure provides antibodies and antibody fragments thatbind to GFRAL. In certain circumstances there are advantages of usingantibody fragments, rather than whole antibodies. The smaller size ofthe fragments allows for rapid clearance, and may lead to improvedaccess to cells, tissues or organs. For a review of certain antibodyfragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli or yeastcells, thus allowing the facile production of large amounts of thesefragments. Antibody fragments can be isolated from the antibody phagelibraries discussed above. Alternatively, Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)₂fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Accordingto another approach, F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Fab and F(ab′)₂ fragment with increasedin vivo half-life comprising salvage receptor binding epitope residuesare described, for example, U.S. Pat. No. 5,869,046. Other techniquesfor the production of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458). Fv and scFv have intact combining sites that are devoid ofconstant regions; thus, they may be suitable for reduced nonspecificbinding during in vivo use. scFv fusion proteins may be constructed toyield fusion of an effector protein at either the amino or the carboxyterminus of an scFv. (See, e.g., Antibody Engineering, ed. Borrebaeck,supra). The antibody fragment may also be a “linear antibody”, forexample, as described, for example, in the references cited above. Suchlinear antibodies may be monospecific or multi-specific, such asbispecific.

Smaller antibody-derived binding structures are the separate variabledomains (V domains) also termed single variable domain antibodies(SdAbs). Certain types of organisms, the camelids and cartilaginousfish, possess high affinity single V-like domains mounted on an Fcequivalent domain structure as part of their immune system. (Woolven etal., Immunogenetics 50: 98-101, 1999; Streltsov et al., Proc Natl AcadSci USA. 101:12444-12449, 2004). The V-like domains (called VhH incamelids and V-NAR in sharks) typically display long surface loops,which allow penetration of cavities of target antigens. They alsostabilize isolated VH domains by masking hydrophobic surface patches.

These VhH and V-NAR domains have been used to engineer sdAbs. Human Vdomain variants have been designed using selection from phage librariesand other approaches that have resulted in stable, high binding VL- andVH-derived domains.

Antibodies that bind to GFRAL as provided herein include, but are notlimited to, synthetic antibodies, monoclonal antibodies, recombinantlyproduced antibodies, multispecific antibodies (including bi-specificantibodies), human antibodies, humanized antibodies, camelizedantibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id)antibodies, and functional fragments, (e.g., GFRAL binding fragments) ofany of the above. Non-limiting examples of functional fragments (e.g.,fragments that bind to GFRAL) include single-chain Fvs (scFv) (e.g.,including monospecific, bispecific, etc.), Fab fragments, F(ab′)fragments, F(ab)2 fragements, F(ab′)2 fragments, disulfide-linked Fvs(sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody andminibody.

Antibodies provided herein include, but are not limited to,immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, for example, molecules that contain an antigenbinding site that bind to a GFRAL epitope. The immunoglobulin moleculesprovided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

Variants and derivatives of antibodies include antibody functionalfragments that retain the ability to bind to a GFRAL epitope. Exemplaryfunctional fragments include Fab fragments (e.g., an antibody fragmentthat contains the antigen-binding domain and comprises a light chain andpart of a heavy chain bridged by a disulfide bond); Fab′ (e.g., anantibody fragment containing a single anti-binding domain comprising anFab and an additional portion of the heavy chain through the hingeregion); F(ab′)2 (e.g., two Fab' molecules joined by interchaindisulfide bonds in the hinge regions of the heavy chains; the Fab'molecules may be directed toward the same or different epitopes); abispecific Fab (e.g., a Fab molecule having two antigen binding domains,each of which may be directed to a different epitope); a single chainFab chain comprising a variable region, also known as, a sFv (e.g., thevariable, antigen-binding determinative region of a single light andheavy chain of an antibody linked together by a chain of 10-25 aminoacids); a disulfide-linked Fv, or dsFv (e.g., the variable,antigen-binding determinative region of a single light and heavy chainof an antibody linked together by a disulfide bond); a camelized VH(e.g., the variable, antigen-binding determinative region of a singleheavy chain of an antibody in which some amino acids at the VH interfaceare those found in the heavy chain of naturally occurring camelantibodies); a bispecific sFv (e.g., a sFv or a dsFv molecule having twoantigen-binding domains, each of which may be directed to a differentepitope); a diabody (e.g., a dimerized sFv formed when the VH domain ofa first sFv assembles with the VL domain of a second sFv and the VLdomain of the first sFv assembles with the VH domain of the second sFv;the two antigen-binding regions of the diabody may be directed towardsthe same or different epitopes); and a triabody (e.g., a trimerized sFv,formed in a manner similar to a diabody, but in which threeantigen-binding domains are created in a single complex; the threeantigen binding domains may be directed towards the same or differentepitopes). Derivatives of antibodies also include one or more CDRsequences of an antibody combining site. The CDR sequences may be linkedtogether on a scaffold when two or more CDR sequences are present. Incertain embodiments, the antibody comprises a single-chain Fv (“scFv”).scFvs are antibody fragments comprising the VH and VL domains of anantibody, wherein these domains are present in a single polypeptidechain. The scFv polypeptide may further comprise a polypeptide linkerbetween the VH and VL domains which enables the scFv to form the desiredstructure for antigen binding. For a review of scFvs see Pluckthun inThe Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, N.Y., pp. 269-315 (1994).

4. Humanized Antibodies

The present disclosure provides humanized antibodies that bind GFRAL,including human GFRAL. Humanized antibodies of the present disclosuremay comprise one or more CDRs as shown in Tables 1-24. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source that is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization may be performed,for example, following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody.

In some cases, the humanized antibodies are constructed by CDR grafting,in which the amino acid sequences of the six complementarity determiningregions (CDRs) of the parent non-human antibody (e.g., rodent) aregrafted onto a human antibody framework. For example, Padlan et al.(FASEB J. 9:133-139, 1995) determined that only about one third of theresidues in the CDRs actually contact the antigen, and termed these the“specificity determining residues,” or SDRs. In the technique of SDRgrafting, only the SDR residues are grafted onto the human antibodyframework (see, e.g., Kashmiri et al., Methods 36: 25-34, 2005).

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. For example, according to the so-called “best-fit” method,the sequence of the variable domain of a non-human (e.g., rodent)antibody is screened against the entire library of known humanvariable-domain sequences. The human sequence which is closest to thatof the rodent may be selected as the human framework for the humanizedantibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987)J. Mol. Biol. 196:901. Another method uses a particular frameworkderived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains. The same framework may beused for several different humanized antibodies (Carter et al. (1992)Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol.,151:2623. In some cases, the framework is derived from the consensussequences of the most abundant human subclasses, V_(L)6 subgroup I(V_(L)6I) and VH subgroup III (V_(H)III). In another method, humangermline genes are used at the source of the framework regions.

In an alternative paradigm based on comparison of CDRs, calledSuperhumanization, FR homology is irrelevant. The method consists ofcomparison of the non-human sequence with the functional human germ linegene repertoire. Those genes encoding the same or closely relatedcanonical structures to the murine sequences are then selected. Next,within the genes sharing the canonical structures with the non-humanantibody, those with highest homology within the CDRs are chosen as FRdonors. Finally, the non-human CDRs are grafted onto these FRs (see,e.g., Tan et al., J. Immunol. 169: 1119-1125, 2002).

It is further generally desirable that antibodies be humanized withretention of their affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Theseinclude, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824,2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993),and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713,1997). Inspection of these displays permits analysis of the likely roleof the residues in the functioning of the candidate immunoglobulinsequence, e.g., the analysis of residues that influence the ability ofthe candidate immunoglobulin to bind its antigen. In this way, FRresidues can be selected and combined from the recipient and importsequences so that the desired antibody characteristic, such as increasedaffinity for the target antigen(s), is achieved. In general, thehypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Another method for antibody humanization is based on a metric ofantibody humanness termed Human String Content (HSC). This methodcompares the mouse sequence with the repertoire of human germ line genesand the differences are scored as HSC. The target sequence is thenhumanized by maximizing its HSC rather than using a global identitymeasure to generate multiple diverse humanized variants. (Lazar et al.,Mol. Immunol. 44: 1986-1998, 2007).

In addition to the methods described above, empirical methods may beused to generate and select humanized antibodies. These methods includethose that are based upon the generation of large libraries of humanizedvariants and selection of the best clones using enrichment technologiesor high throughput screening techniques. Antibody variants may beisolated from phage, ribosome and yeast display libraries as well as bybacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006;Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al.,Protein Eng. Des. Sel. 17: 847-60, 2004).

In the FR library approach, a collection of residue variants areintroduced at specific positions in the FR followed by selection of thelibrary to select the FR that best supports the grafted CDR. Theresidues to be substituted may include some or all of the “Vernier”residues identified as potentially contributing to CDR structure (see,e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from themore limited set of target residues identified by Baca et al. (J. Biol.Chem. 272: 10678-10684, 1997).

In FR shuffling, whole FRs are combined with the non-human CDRs insteadof creating combinatorial libraries of selected residue variants (see,e.g., Dall'Acqua et al., Methods 36: 43-60, 2005). The libraries may bescreened for binding in a two-step selection process, first humanizingVL, followed by VH. Alternatively, a one-step FR shuffling process maybe used. Such a process has been shown to be more efficient than thetwo-step screening, as the resulting antibodies exhibited improvedbiochemical and physico-chemical properties including enhancedexpression, increased affinity and thermal stability (see, e.g.,Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

The “humaneering” method is based on experimental identification ofessential minimum specificity determinants (MSDs) and is based onsequential replacement of non-human fragments into libraries of humanFRs and assessment of binding. It begins with regions of the CDR3 ofnon-human VH and VL chains and progressively replaces other regions ofthe non-human antibody into the human FRs, including the CDR1 and CDR2of both VH and VL. This methodology typically results in epitoperetention and identification of antibodies from multiple sub-classeswith distinct human V-segment CDRs. Humaneering allows for isolation ofantibodies that are 91-96% homologous to human germline gene antibodies.(see, e.g., Alfenito, Cambridge Healthtech Institute's Third AnnualPEGS, The Protein Engineering Summit, 2007).

The “human engineering” method involves altering an non-human antibodyor antibody fragment, such as a mouse or chimeric antibody or antibodyfragment, by making specific changes to the amino acid sequence of theantibody so as to produce a modified antibody with reducedimmunogenicity in a human that nonetheless retains the desirable bindingproperties of the original non-human antibodies. Generally, thetechnique involves classifying amino acid residues of a non-human (e.g.,mouse) antibody as “low risk”, “moderate risk”, or “high risk” residues.The classification is performed using a global risk/reward calculationthat evaluates the predicted benefits of making particular substitution(e.g., for immunogenicity in humans) against the risk that thesubstitution will affect the resulting antibody's folding and/or aresubstituted with human residues. The particular human amino acid residueto be substituted at a given position (e.g., low or moderate risk) of anon-human (e.g., mouse) antibody sequence can be selected by aligning anamino acid sequence from the non-human antibody's variable regions withthe corresponding region of a specific or consensus human antibodysequence. The amino acid residues at low or moderate risk positions inthe non-human sequence can be substituted for the corresponding residuesin the human antibody sequence according to the alignment. Techniquesfor making human engineered proteins are described in greater detail inStudnicka et al., Protein Engineering, 7: 805-814 (1994), U.S. Pat. Nos.5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT ApplicationPublication WO 93/11794.

5. Human Antibodies

Human anti-GFRAL antibodies can be constructed by combining Fv clonevariable domain sequence(s) selected from human-derived phage displaylibraries with known human constant domain sequences(s). Alternatively,human monoclonal GFRAL antibodies of the present disclosure can be madeby the hybridoma method. Human myeloma and mouse-human heteromyelomacell lines for the production of human monoclonal antibodies have beendescribed, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., N.Y., 1987); and Boerner et al., J.Immunol., 147: 86 (1991).

It is also possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Transgenic mice that express human antibody repertoires have been usedto generate high-affinity human sequence monoclonal antibodies against awide variety of potential drug targets (see, e.g., Jakobovits, A., Curr.Opin. Biotechnol. 1995, 6(5):561-6; Brüggemann and Taussing, Curr. Opin.Biotechnol. 1997, 8(4):455-8; U.S. Pat. Nos. 6,075,181 and 6,150,584;and Lonberg et al., Nature Biotechnol. 23: 1117-1125, 2005).

Alternatively, the human antibody may be prepared via immortalization ofhuman B lymphocytes producing an antibody directed against a targetantigen (e.g., such B lymphocytes may be recovered from an individual ormay have been immunized in vitro) (see, e.g., Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373).

Gene shuffling can also be used to derive human antibodies fromnon-human, for example, rodent, antibodies, where the human antibody hassimilar affinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting” or“guided selection”, either the heavy or light chain variable region of anon-human antibody fragment obtained by phage display techniques asdescribed herein is replaced with a repertoire of human V domain genes,creating a population of non-human chain/human chain scFv or Fabchimeras. Selection with antigen results in isolation of a non-humanchain/human chain chimeric scFv or Fab wherein the human chain restoresthe antigen binding site destroyed upon removal of the correspondingnon-human chain in the primary phage display clone, (e.g., the epitopeguides (imprints) the choice of the human chain partner). When theprocess is repeated in order to replace the remaining non-human chain, ahuman antibody is obtained (see, e.g., PCT WO 93/06213; and Osbourn etal., Methods., 36, 61-68, 2005). Unlike traditional humanization ofnon-human antibodies by CDR grafting, this technique provides completelyhuman antibodies, which have no FR or CDR residues of non-human origin.Examples of guided selection to humanize mouse antibodies towards cellsurface antigens include the folate-binding protein present on ovariancancer cells (see, e.g., Figini et al., Cancer Res., 58, 991-996, 1998)and CD147, which is highly expressed on hepatocellular carcinoma (see,e.g., Bao et al., Cancer Biol. Ther., 4, 1374-1380, 2005).

A potential disadvantage of the guided selection approach is thatshuffling of one antibody chain while keeping the other constant couldresult in epitope drift. In order to maintain the epitope recognized bythe non-human antibody, CDR retention can be applied (see, e.g., Klimkaet al., Br. J. Cancer., 83, 252-260, 2000; VH CDR2 Beiboer et al., J.Mol. Biol., 296, 833-49, 2000) In this method, the non-human VH CDR3 iscommonly retained, as this CDR may be at the center of theantigen-binding site and may be to be the most important region of theantibody for antigen recognition. In some instances, however, VH CDR3and VL CDR3, as well as VH CDR3, VL CDR3 and VL CFR1, of the non-humanantibody may be retained.

6. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for GFRAL andthe other is for any other antigen. In some embodiments, bispecificantibodies can bind to two different epitopes of GFRAL. Bispecificantibodies can be prepared as full length antibodies or antibodyfragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art, such as,for example, by co-expression of two immunoglobulin heavy chain-lightchain pairs, where the two heavy chains have different specificities(see, e.g., Milstein and Cuello, Nature, 305: 537 (1983)). For furtherdetails of generating bispecific antibodies see, for example, BispecificAntibodies, Kontermann, ed., Springer-Verlag, Hiedelberg (2011).

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present disclosure can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g., tetravalent antibodies),which can be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, amultivalent antibody comprises (or consists of) three to about eightantigen binding sites. In one such embodiment, a multivalent antibodycomprises (or consists of) four antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (for example, twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

8. Fc Engineering

It may be desirable to modify an antibody to GFRAL via Fc engineering,including, with respect to effector function, for example, so as todecrease or remove antigen-dependent cell-mediated cyotoxicity (ADCC)and/or complement dependent cytotoxicity (CDC) of the antibody. This maybe achieved by introducing one or more amino acid substitutions in an Fcregion of the antibody. For example, substitutions into human IgG1 usingIgG2 residues as positions 233-236 and IgG4 residues at positions 327,330 and 331 were shown to greatly reduce ADCC and CDC (see, e.g., Armouret al., Eur. J. Immunol. 29:(8):2613-24 (1999); Shields et al., J. Biol.Chem. 276(9): 6591-604 (2001).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment), for example, as described in U.S. Pat. No.5,739,277. Term “salvage receptor binding epitope” refers to an epitopeof the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the in vivo serum half-life of theIgG molecule.

9. Alternative Binding Agents

The present disclosure encompasses non-immunoglobulin binding agentsthat specifically bind to the same epitope as an anti-GFRAL antibodydisclosed herein. In some embodiments, a non-immunoglobulin bindingagent is identified an agent that displaces or is displaced by ananti-GFRAL antibody of the present disclosure in a competive bindingassay. These alternative binding agents may include, for example, any ofthe engineered protein scaffolds known in the art. Such scaffolds maycomprise one or more CDRs as shown in Tables 1-24. Such scaffoldsinclude, for example, anticalins, which are based upon the lipocalinscaffold, a protein structure characterized by a rigid beta-barrel thatsupports four hypervariable loops which form the ligand binding site.Novel binding specificities may be engineered by targeted randommutagenesis in the loop regions, in combination with functional displayand guided selection (see, e.g., Skerra (2008) FEBS J. 275: 2677-2683).Other suitable scaffolds may include, for example, adnectins, ormonobodies, based on the tenth extracellular domain of human fibronectinIII (see, e.g., Koide and Koide (2007) Methods Mol. Biol. 352: 95-109);affibodies, based on the Z domain of staphylococcal protein A (see,e.g., Nygren et al. (2008) FEBS J. 275: 2668-2676)); DARPins, based onankyrin repeat proteins (see, e.g., Stumpp et al. (2008) Drug. Discov.Today 13: 695-701); fynomers, based on the SH3 domain of the human Fynprotein kinase Grabulovski et al. (2007) J. Biol. Chem. 282: 3196-3204);affitins, based on Sac7d from Sulfolobus acidolarius (see, e.g.,Krehenbrink et al. (2008) J. Mol. Biol. 383: 1058-1068); affilins, basedon human y-B-crystallin (see, e.g., Ebersbach et al. (2007) J. Mol.Biol. 372: 172-185); avimers, based on the A domains of membranereceptor proteins (see, e.g., Silverman et al. (2005) Biotechnol. 23:1556-1561); cysteine-rich knottin peptides (see, e.g., Kolmar (2008)FEBS J. 275: 2684-2690); and engineered Kunitz-type inhibitors (see,e.g., Nixon and Wood (2006) Curr. Opin. Drug. Discov. Dev. 9: 261-268)For a review, see, for example, Gebauer and Skerra (2009) Curr. Opin.Chem. Biol. 13: 245-255.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies that bind to GFRAL described herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody, including but not limitedto specificity, thermostability, expression level, effector functions,glycosylation, reduced immunogenicity or solubility. This, in additionto the anti-GFRAL antibodies described herein, it is contemplated thatanti-GFRAL antibody variants can be prepared. For example, anti-GFRALantibody variants can be prepared by introducing appropriate nucleotidechanges into the encoding DNA, and/or by synthesis of the desiredantibody or polypeptide. Those skilled in the art will appreciate thatamino acid changes may alter post-translational processes of theanti-GFRAL antibody, such as changing the number or position ofglycosylation sites or altering the membrane anchoring characteristics.

In some embodiments, antibodies provided herein are chemically modified,for example, by the association with, including covalent attachment of,any type of molecule with the antibody. The antibody derivatives mayinclude antibodies that have been chemically modified, for example, byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited, to specific chemical cleavage, acetylation,formulation, metabolic synthesis of tunicamycin, etc. Additionally, theantibody may contain one or more non-classical amino acids.

Variations may be a substitution, deletion or insertion of one or morecodons encoding the antibody or polypeptide that results in a change inthe amino acid sequence as compared with the native sequence antibody orpolypeptide. Amino acid substitutions can be the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,e.g., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids. In certainembodiments, the substitution, deletion or insertion includes less than25 amino acid substitutions, less than 20 amino acid substitutions, lessthan 15 amino acid substitutions, less than 10 amino acid substitutions,less than 5 amino acid substitutions, less than 4 amino acidsubstitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the original molecule. In aspecific embodiment, the substitution is a conservative amino acidsubstitution made at one or more predicted non-essential amino acidresidues. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for antibody-directedenzyme prodrug therapy) or a polypeptide which increases the serumhalf-life of the antibody.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Alternatively,conservative (e.g., within an amino acid group with similar propertiesand/or sidechains) substitutions may be made, so as to maintain or notsignificantly change the properties. Amino acids may be groupedaccording to similarities in the properties of their side chains (see,e.g., A. L. Lehninger, in Biochemistry, 2nd Ed., pp. 73-75, WorthPublishers, N.Y. (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile(I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G),Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp(D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H).

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites. Accordingly, in one embodiment, anantibody or fragment thereof that binds to a GFRAL epitope comprises anamino acid sequence that is at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the amino acid sequence of a murine monoclonalantibody described herein. In one embodiment, an antibody or fragmentthereof that binds to a GFRAL epitope comprises an amino acid sequencethat is at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99% identicalto an amino acid sequence depicted in Tables 1-24. In yet anotherembodiment, an antibody or fragment thereof that binds to a GFRALepitope comprises a VH CDR and/or a VL CDR amino acid sequence that isat least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical to a VHCDR amino acid sequence depicted in Tables 1-24 and/or a VL CDR aminoacid sequence depicted in Tables 1-24. The variations can be made usingmethods known in the art such as oligonucleotide-mediated(site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res.,13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)),cassette mutagenesis (see, e.g., Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniquescan be performed on the cloned DNA to produce the anti-GFRAL antibodyvariant DNA.

Any cysteine residue not involved in maintaining the proper conformationof the anti-GFRAL antibody also may be substituted, for example, withwith another amino acid such as alanine or serine, to improve theoxidative stability of the molecule and prevent aberrant crosslinking.Conversely, cysteine bond(s) may be added to the anti-GFRAL antibody toimprove its stability (e.g., where the antibody is an antibody fragmentsuch as an Fv fragment).

In some embodiments, an anti-GFRAL antibody molecule of the presentdisclosure is a “de-immunized” antibody. A “de-immunized” anti-GFRALantibody is an antibody derived from a humanized or chimeric anti-GFRALantibody, that has one or more alterations in its amino acid sequenceresulting in a reduction of immunogenicity of the antibody, compared tothe respective original non-de-immunized antibody. One of the proceduresfor generating such antibody mutants involves the identification andremoval of T-cell epitopes of the antibody molecule. In a first step,the immunogenicity of the antibody molecule can be determined by severalmethods, for example, by in vitro determination of T-cell epitopes or insilico prediction of such epitopes, as known in the art. Once thecritical residues for T-cell epitope function have been identified,mutations can be made to remove immunogenicity and retain antibodyactivity. For review, see, for example, Jones et al., Methods inMolecular Biology 525: 405-423, 2009.

1. In vitro Affinity Maturation

In some embodiments, antibody variants having an improved property suchas affinity, stability, or expression level as compared to a parentantibody may be prepared by in vitro affinity maturation. Like thenatural prototype, in vitro affinity maturation is based on theprinciples of mutation and selection. Libraries of antibodies aredisplayed as Fab, scFv or V domain fragments either on the surface of anorganism (e.g., phage, bacteria, yeast or mammalian cell) or inassociation (e.g., covalently or non-covalently) with their encodingmRNA or DNA. Affinity selection of the displayed antibodies allowsisolation of organisms or complexes carrying the genetic informationencoding the antibodies. Two or three rounds of mutation and selectionusing display methods such as phage display usually results in antibodyfragments with affinities in the low nanomolar range. Preferred affinitymatured antibodies will have nanomolar or even picomolar affinities forthe target antigen.

Phage display is a widepread method for display and selection ofantibodies. The antibodies are displayed on the surface of Fd or M13bacteriophages as fusions to the bacteriophage coat protein. Selectioninvolves exposure to antigen to allow phage-displayed antibodies to bindtheir targets, a process referred to as “panning.” Phage bound toantigen are recovered and infected in bacteria to produce phage forfurther rounds of selection. For review, see, for example, Hoogenboom,Methods. Mol. Biol. 178: 1-37, 2002; Bradbury and Marks, J. Immuno.Methods 290: 29-49, 2004).

In a yeast display system (see, e.g., Boder et al., Nat. Biotech. 15:553-57, 1997; Chao et al., Nat. Protocols 1:755-768, 2006), the antibodymay be displayed as single-chain variable fusions (scFv) in which theheavy and light chains are connected by a flexible linker. The scFv isfused to the adhesion subunit of the yeast agglutinin protein Aga2p,which attaches to the yeast cell wall through disulfide bonds to Aga1p.Display of a protein via Aga2p projects the protein away from the cellsurface, minimizing potential interactions with other molecules on theyeast cell wall. Magnetic separation and flow cytometry are used toscreen the library to select for antibodies with improved affinity orstability. Binding to a soluble antigen of interest is determined bylabeling of yeast with biotinylated antigen and a secondary reagent suchas streptavidin conjugated to a fluorophore. Variations in surfaceexpression of the antibody can be measured through immunofluorescencelabeling of either the hemagglutinin or c-Myc epitope tag flanking thescFv. Expression has been shown to correlate with the stability of thedisplayed protein, and thus antibodies can be selected for improvedstability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol.292: 949-956, 1999). An additional advantage of yeast display is thatdisplayed proteins are folded in the endoplasmic reticulum of theeukaryotic yeast cells, taking advantage of endoplasmic reticulumchaperones and quality-control machinery. Once maturation is complete,antibody affinity can be conveniently ‘titrated’ while displayed on thesurface of the yeast, eliminating the need for expression andpurification of each clone. A theoretical limitation of yeast surfacedisplay is the potentially smaller functional library size than that ofother display methods; however, a recent approach uses the yeast cells'mating system to create combinatorial diversity estimated to be 10¹⁴ insize (see, e.g., US Patent Publication 2003/0186,374; Blaise et al.,Gene 342: 211-218, 2004).

In ribosome display, antibody-ribosome-mRNA (ARM) complexes aregenerated for selection in a cell-free system. The DNA library codingfor a particular library of antibodies is genetically fused to a spacersequence lacking a stop codon. This spacer sequence, when translated, isstill attached to the peptidyl tRNA and occupies the ribosomal tunnel,and thus allows the protein of interest to protrude out of the ribosomeand fold. The resulting complex of mRNA, ribosome, and protein can bindto surface-bound ligand, allowing simultaneous isolation of the antibodyand its encoding mRNA through affinity capture with the ligand. Theribosome-bound mRNA is then reversed transcribed back into cDNA, whichcan then undergo mutagenesis and be used in the next round of selection(see, e.g., Fukuda et al., Nucleic Acids Res. 34, e127, 2006). In mRNAdisplay, a covalent bond between antibody and mRNA is established usingpuromycin as an adaptor molecule (Wilson et al., Proc. Natl. Acad. Sci.USA 98, 3750-3755, 2001).

As these methods are performed entirely in vitro, they provide two mainadvantages over other selection technologies. First, the diversity ofthe library is not limited by the transformation efficiency of bacterialcells, but only by the number of ribosomes and different mRNA moleculespresent in the test tube. Second, random mutations can be introducedeasily after each selection round, for example, by non-proofreadingpolymerases, as no library must be transformed after any diversificationstep.

Diversity may be introduced into the CDRs or the whole V genes of theantibody libraries in a targeted manner or via random introduction. Theformer approach includes sequentially targeting all the CDRs of anantibody via a high or low level of mutagenesis or targeting isolatedhot spots of somatic hypermutations (see, e.g., Ho, et al., J. Biol.Chem. 280: 607-617, 2005) or residues suspected of affecting affinity onexperimental basis or structural reasons. Random mutations can beintroduced throughout the whole V gene using E. coli mutator strains,error-prone replication with DNA polymerases (see, e.g., Hawkins et al.,J. Mol. Biol. 226: 889-896, 1992) or RNA replicases. Diversity may alsobe introduced by replacement of regions that are naturally diverse viaDNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol.Chem. 278: 43496-43507, 2003; U.S. Pat. No. 5,565,332; U.S. Pat. No.6,989,250). Alternative techniques target hypervariable loops extendinginto framework-region residues (see, e.g., Bond et al., J. Mol. Biol.348: 699-709, 2005) employ loop deletions and insertions in CDRs or usehybridization-based diversification (see, e.g., US Patent PublicationNo. 2004/0005709). Additional methods of generating diversity in CDRsare disclosed, for example, in U.S. Pat. No. 7,985,840.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, GFRAL can be immobilized onto solidsupports, columns, pins or cellulose/poly(vinylidene fluoride)membranes/ other filters, expressed on host cells affixed to adsorptionplates or used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panningdisplay libraries.

For review of in vitro affinity maturation methods, see, e.g.,Hoogenboom, Nature Biotechnology 23: 1105-1116, 2005 and Quiroz andSinclair, Revista Ingeneria Biomedia 4: 39-51, 2010 and referencestherein.

2. Modifications of Anti-GFRAL Antibodies

Covalent modifications of anti-GFRAL antibodies are included within thescope of the present disclosure. Covalent modifications include reactingtargeted amino acid residues of an anti-GFRAL antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C- terminal residues of the anti-GFRAL antibody. Othermodifications include deamidation of glutaminyl and asparaginyl residuesto the corresponding glutamyl and aspartyl residues, respectively,hydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the α-amino groups oflysine, arginine, and histidine side chains (see, e.g., T. E. Creighton,Proteins: Structure and Molecular Properties, W. H. Freeman & Co., SanFrancisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, andamidation of any C-terminal carboxyl group.

Other types of covalent modification of the anti-GFRAL antibody includedwithin the scope of this present disclosure include altering the nativeglycosylation pattern of the antibody or polypeptide (see, e.g., Beck etal., Curr. Pharm. Biotechnol. 9: 482-501, 2008; Walsh, Drug Discov.Today 15: 773-780, 2010), and linking the antibody to one of a varietyof nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth, forexample, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337.

An anti-GFRAL antibody of the present disclosure may also be modified toform chimeric molecules comprising an anti-GFRAL antibody fused toanother, heterologous polypeptide or amino acid sequence, for example,an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-533, 2003) or the Fc region of an IgG molecule (see, e.g., Aruffo,“Immunoglobulin fusion proteins” in Antibody Fusion Proteins, S. M.Chamow and A. Ashkenazi, eds., Wiley-Liss, N.Y., 1999, pp. 221-242).

Also provided herein are fusion proteins comprising an antibody providedherein that binds to a GFRAL antigen and a heterologous polypeptide. Insome embodiments, the heterologous polypeptide to which the antibody isfused is useful for targeting the antibody to cells having cellsurface-expressed GFRAL.

Also provided herein are panels of antibodies that bind to a GFRALantigen. In specific embodiments, panels of antibodies have differentassociation rate constants different dissociation rate constants,different affinities for GFRAL antigen, and/or different specificitiesfor a GFRAL antigen. In some embodiments, the panels comprise or consistof about 10, about 25, about 50, about 75, about 100, about 125, about150, about 175, about 200, about 250, about 300, about 350, about 400,about 450, about 500, about 550, about 600, about 650, about 700, about750, about 800, about 850, about 900, about 950, or about 1000antibodies or more. Panels of antibodies can be used, for example, in 96well or 384 well plates, such as for assays such as ELISAs.

Preparation of Anti-GFRAL Antibodies

Anti-GFRAL antibodies may be produced by culturing cells transformed ortransfected with a vector containing anti-GFRAL antibody-encodingnucleic acids. Polynucleotide sequences encoding polypeptide componentsof the antibody of the present disclosure can be obtained using standardrecombinant techniques. Desired polynucleotide sequences may be isolatedand sequenced from antibody producing cells such as hybridomas cells.Alternatively, polynucleotides can be synthesized using nucleotidesynthesizer or PCR techniques. Once obtained, sequences encoding thepolypeptides are inserted into a recombinant vector capable ofreplicating and expressing heterologous polynucleotides in host cells.Many vectors that are available and known in the art can be used for thepurpose of the present disclosure. Selection of an appropriate vectorwill depend mainly on the size of the nucleic acids to be inserted intothe vector and the particular host cell to be transformed with thevector. Host cells suitable for expressing antibodies of the presentdisclosure include prokaryotes such as Archaebacteria and Eubacteria,including Gram-negative or Gram-positive organisms, eukaryotic microbessuch as filamentous fungi or yeast, invertebrate cells such as insect orplant cells, and vertebrate cells such as mammalian host cell lines.Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Antibodies produced by the host cellsare purified using standard protein purification methods as known in theart.

Methods for antibody production including vector construction,expression and purification are further described, in Plückthun et al.,(1996) in Antibody Engineering: Producing antibodies in Escherichiacoli: From PCR to fermentation (McCafferty, J., Hoogenboom, H. R., andChiswell, D. J., eds), 1 Ed., pp. 203-252, IRL Press, Oxford; Kwong, K.& Rader, C., E. coli expression and purification of Fab antibodyfragments, Current protocols in protein science editorial board John EColigan et al., Chapter 6, Unit 6.10 (2009); Tachibana and Takekoshi,“Production of Antibody Fab Fragments in Escherischia coli,” in AntibodyExpression and Production, M. Al-Rubeai, Ed., Springer, N.Y., 2011;Therapeutic Monoclonal Antibodies: From Bench to Clinic (ed Z. An), JohnWiley & Sons, Inc., Hoboken, N.J., USA.

It is, of course, contemplated that alternative methods, which are wellknown in the art, may be employed to prepare anti-GFRAL antibodies. Forinstance, the appropriate amino acid sequence, or portions thereof, maybe produced by direct peptide synthesis using solid-phase techniques(see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W. H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)). In vitro protein synthesis may be performed usingmanual techniques or by automation. Various portions of the anti-GFRALantibody may be chemically synthesized separately and combined usingchemical or enzymatic methods to produce the desired anti-GFRALantibody. Alternatively, antibodies may be purified from cells or bodilyfluids, such as milk, of a transgenic animal engineered to express theantibody, as disclosed, for example, in U.S. Pat. No. 5,545,807 and U.S.Pat. No. 5,827,690.

Immunoconiugates

The present disclosure also provides conjugates comprising any one ofthe anti-GFRAL antibodies of the present disclosure covalently bound,including by a synthetic linker, to one or more non-antibody agents.

A variety of radioactive isotopes are available for the production ofradioconjugated antibodies. Examples include At²¹¹, I4, I4, Y4, Re4,Re4, Sm4, Bi4, P4, Pb4 and radioactive isotopes of Lu. When theconjugate is used for detection, it may comprise a radioactive atom forscintigraphic studies, for example tc4 or I4, or a spin label fornuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, MRI), such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron. The radioisotopes may be incorporated in theconjugate in known ways as described, e.g., in Reilly, “Theradiochemistry of monoclonal antibodies and peptides,” in MonoclonalAntibody and Peptide-Targeted Radiotherapy of Cancer, R. M. Reilly, ed.,Wiley, Hoboken N.J., 2010.

In some embodiments, antibodies provided herein are conjugated orrecombinantly fused to a diagnostic, detectable or therapeutic agent orany other molecule. The conjugated or recombinantly fused antibodies canbe useful, for example, for monitoring or prognosing the onset,development, progression and/or severity of a β-cell defective disease,disorder or condition as part of a clinical testing procedure, such asdetermining the efficacy of a particular therapy.

Such diagnosis and detection can accomplished, for example, by couplingthe antibody to detectable substances including, but not limited to,various enzymes, such as, but not limited to, horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;prosthetic groups, such as, but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials, such as, but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as, but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; chemiluminescent material, such as but notlimited to, an acridinium based compound or a HALOTAG; radioactivematerials, such as, but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and¹²¹I,), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In,¹¹²In, and ¹¹¹In,), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga,⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine(¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc,¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd,¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metalsusing various positron emission tomographies, and non-radioactiveparamagnetic metal ions.

Also provided herein are antibodies that are conjugated or recombinantlyfused to a therapeutic moiety (or one or more therapeutic moieties), aswell as uses thereof. The antibody may be conjugated or recombinantlyfused to a therapeutic moiety, including a cytotoxin such as acytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion such as alpha-emitters. A cytotoxin or cytotoxic agentincludes any agent that is detrimental to cells.

Further, an antibody provided herein may be conjugated or recombinantlyfused to a therapeutic moiety or drug moiety that modifies a givenbiological response. Therapeutic moieties or drug moieties are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein, peptide, or polypeptidepossessing a desired biological activity. Such proteins may include, forexample, a toxin such as abrin, ricin A, pseudomonas exotoxin, choleratoxin, or diphtheria toxin; a protein such as tumor necrosis factor,γ-interferon, α-interferon, nerve growth factor, platelet derived growthfactor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-γ,TNF-65 , AIM I (see, e.g., International Publication No. WO 97/33899),AIM II (see, e.g., International Publication No. WO 97/34911), FasLigand (see, e.g.,Takahashi et al., 1994, J. Immunol., 6:1567-1574), andVEGF (see, e.g., International Publication No. WO 99/23105), ananti-angiogenic agent, including, for example angiostatin, endostatin ora component of the coagulation pathway (e.g., tissue factor); or, abiological response modifier such as, for example, a lymphokine (e.g.,interferon gamma, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-5 (“IL-5”), interleukin-6 (“IL-6”), interleukin-7 (“IL-7”),interleukin 9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12(“IL-12”), interleukin-15 (“IL-15”), interleukin-23 (“IL-23”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)) , or a growth factor(e.g., growth hormone (“GH”)), or a coagulation agent (e.g., calcium,vitamin K, tissue factors, such as but not limited to, Hageman factor(factor XII), high-molecular-weight kininogen (HMWK), prekallikrein(PK), coagulation proteins-factors II (prothrombin), factor V, XIIa,VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, and fibrin monomer).

Also provided herein are antibodies that are recombinantly fused orchemically conjugated (covalent or non-covalent conjugations) to aheterologous protein or polypeptide (or fragment thereof, for example,to a polypeptide of about 10, about 20, about 30, about 40, about 50,about 60, about 70, about 80, about 90 or about 100 amino acids) togenerate fusion proteins, as well as uses thereof. In particular,provided herein are fusion proteins comprising an antigen-bindingfragment of an antibody provided herein (e.g., a Fab fragment, Fdfragment, Fv fragment, F(ab)₂ fragment, a VH domain, a VH CDR, a VLdomain or a VL CDR) and a heterologous protein, polypeptide, or peptide.In one embodiment, the heterologous protein, polypeptide, or peptidethat the antibody is fused to is useful for targeting the antibody to aparticular cell type, such as a cell that expresses GFRAL. For example,an antibody that binds to a cell surface receptor expressed by aparticular cell type (e.g., an immune cell) may be fused or conjugatedto a modified antibody provided herein.

In addition, an antibody provided herein can be conjugated totherapeutic moieties such as a radioactive metal ion, such asalpha-emitters such as ²¹³Bi or macrocyclic chelators useful forconjugating radiometal ions, including but not limited to, ¹³¹In, ¹³¹LU,¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides. In certain embodiments, themacrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described, for example, inDenardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al.,1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl.Med. Biol. 26(8):943-50.

Moreover, antibodies provided herein can be fused to marker or “tag”sequences, such as a peptide to facilitate purification. In specificembodiments, the marker or tag amino acid sequence is a hexa-histidinepeptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN,Inc.), among others, many of which are commercially available. Forexample, as described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA86:821-824, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the hemagglutinin (“HA”) tag, which corresponds toan epitope derived from the influenza hemagglutinin protein (Wilson etal., 1984, Cell 37:767), and the “FLAG” tag.

Methods for fusing or conjugating therapeutic moieties (includingpolypeptides) to antibodies are well known, (see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies 84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol.Rev. 62:119-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813;Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991;Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol.,154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341, 1992).

Fusion proteins may be generated, for example, through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of anti-GFRAL antibodies as providedherein, including, for example, antibodies with higher affinities andlower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997,Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzoand Blasco, 1998, Biotechniques 24(2):308- 313). Antibodies, or theencoded antibodies, may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. A polynucleotide encoding an antibodyprovided herein may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

An antibody provided herein can also be conjugated to a second antibodyto form an antibody heteroconjugate as described, for example, in U.S.Pat. No. 4,676,980.

The therapeutic moiety or drug conjugated or recombinantly fused to anantibody provided herein that binds to GFRAL (e.g., a GFRAL polypeptide,fragment, epitope) should be chosen to achieve the desired prophylacticor therapeutic effect(s). In certain embodiments, the antibody is amodified antibody. A clinician or other medical personnel may consider,for example, the following when deciding on which therapeutic moiety ordrug to conjugate or recombinantly fuse to an antibody provided herein:the nature of the disease, the severity of the disease, and thecondition of the subject.

Antibodies that bind to GFRAL as provided herein may also be attached tosolid supports, which are particularly useful for immunoassays orpurification of the target antigen. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

The linker may be a “cleavable linker” facilitating release of theconjugated agent in the cell, but non-cleavable linkers are alsocontemplated herein. Linkers for use in the conjugates of the presentdisclosure include without limitation acid labile linkers (e.g.,hydrazone linkers), disulfide-containing linkers, peptidase-sensitivelinkers (e.g., peptide linkers comprising amino acids, for example,valine and/or citrulline such as citrulline-valine orphenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g.,Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.5,208,020), thioether linkers, or hydrophilic linkers designed to evademultidrug transporter-mediated resistance (see, e.g., Kovtun et al.,Cancer Res. 70: 2528-2537, 2010).

Conjugates of the antibody and agent may be made using a variety ofbifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS,LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). Thepresent disclosure further contemplates that conjugates of antibodiesand agents may be prepared using any suitable methods as disclosed inthe art, (see, e.g., in Bioconjugate Techniques, 2nd Ed., G.T.Hermanson, ed., Elsevier, San Francisco, 2008).

Conventional conjugation strategies for antibodies and agents have beenbased on random conjugation chemistries involving the ϵ-amino group ofLys residues or the thiol group of Cys residues, which results inheterogenous conjugates. Recently developed techniques allowsite-specific conjugation to antibodies, resulting in homogeneousloading and avoiding conjugate subpopulations with alteredantigen-binding or pharmacokinetics. These include engineering of“thiomabs” comprising cysteine substitutions at positions on the heavyand light chains that provide reactive thiol groups and do not disruptimmunoglobulin folding and assembly or alter antigen binding (see, e.g.,Junutula et al., J. Immunol. Meth. 332: 41-52 (2008); Junutula et al.,Nat. Biotechnol. 26: 925-932, 2008). In another method, selenocysteineis cotranslationally inserted into an antibody sequence by recoding thestop codon UGA from termination to selenocysteine insertion, allowingsite specific covalent conjugation at the nucleophilic selenol group ofselenocysteine in the presence of the other natural amino acids (see,e.g.,Hofer et al., Proc. Natl. Acad. Sci. USA 105: 12451-12456 (2008);Hofer et al., Biochemistry 48(50): 12047-12057, 2009).

Pharmaceutical Formulations

Anti-GFRAL antibodies of the present disclosure may be administered byany route appropriate to the disease, disorder or condition to betreated. The antibody will typically be administered parenterally, forexample, infusion, subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural. The antibody dose will vary,including depending on the nature and/or severity of the disease ordisorder as well as the condition of the subject, may include dosesbetween 1 mg and 100 mg. Doses may also include those between 1 mg/kgand 15 mg/kg. In some embodiments, the dose is between about 5 mg/kg andabout 7.5 mg/kg. In some embodiments, the dose is about 5 mg/kg. In someembodiments, the dose is about 7.5 mg/kg. Flat doses selected from thegroup consisting of: (a) 375-400 mg every two weeks and (b) 550-600 mgevery three weeks. In some embodiments, the flat dose is 375-400 mgevery two weeks. In some embodiments, the flat dose is 550-600 mg everythree weeks. In some embodiments the flat dose is 400 mg every twoweeks. In some embodiments the flat dose is 600 mg every three weeks. Insome embodiments of sequential dosing, a first dose and a second doseare each between 1 mg/kg and 15 mg/kg with the second dose following thefirst does by between 1 and 4 weeks. In some embodiments, the first doseand the second dose are each between 5 mg/kg and 7.5 mg/kg and thesecond dose follows the first dose by between 2 and 3 weeks. In someembodiments, the first dose and the second dose are each 5 mg/kg and thesecond dose follows the first dose by 2 weeks. In some embodiments, thefirst dose and the second dose are each 7.5 mg/kg and the second dosefollows the first dose by 3 weeks.

For treating diseases, disorders or conditions, the antibody in someembodiments is administered via intravenous infusion. The dosageadministered via infusion is in the range of about 1 μg/m² to about10,000 μg/m² per dose, generally one dose per week for a total of one,two, three or four doses. Alternatively, the dosage range is of about 1μg/m² to about 1000 μg/m², about 1 μg/m² to about 800 μg/m², about 1μg/m² to about 600 μg/m², about 1 μg/m² to about 400 μg/m²;alternatively, about 10 μg/m² to about 500 μg/m², about 10 μg/m² toabout 300 μg/m², about 10 μg/m² to about 200 μg/m², and about 1 μg/m² toabout 200 μg/m². The dose may be administered once per day, once perweek, multiple times per week, but less than once per day, multipletimes per month but less than once per day, multiple times per month butless than once per week, once per month or intermittently to relieve oralleviate symptoms of the disease, disorder, or condition.Administration may continue at any of the disclosed intervals untilamelioration of the disease, disorder or condition, or amelioration ofsymptoms of the disease, disorder or condition being treated.Administration may continue after remission or relief of symptoms isachieved where such remission or relief is prolonged by such continuedadministration.

In one aspect, the present disclosure further provides pharmaceuticalformulations comprising at least one anti-GFRAL antibody of the presentdisclosure. In some embodiments, a pharmaceutical formulationcomprises 1) an anti-GFRAL antibody, and 2) a pharmaceuticallyacceptable carrier. In some embodiments, a pharmaceutical formulationcomprises 1) an anti-GFRAL antibody and/or an immunoconjugate thereof,and optionally, 2) at least one additional therapeutic agent.

Pharmaceutical formulations comprising an antibody is prepared forstorage by mixing the antibody having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)) in the form of aqueous solutions or lyophilized or otherdried formulations. The formulations herein may also contain more thanone active compound as necessary for the particular disease, disorder orcondition (e.g., a particular indication) being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to an anti-GFRAL antibody, it may bedesirable to include in the one formulation, an additional antibody,e.g., a second anti-GFRAL antibody which binds a different epitope onthe GFRAL polypeptide, or an antibody to some other target.Alternatively, or additionally, the composition may further compriseanother agent, including, for example, a chemotherapeutic agent,cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent,and/or cardioprotectant. In some embodiments the formulation includes analkylating agent (e.g., chlorambucil, bendamustine hydrochloride orcyclophosphamide) a nucleoside analog (e.g., fludurabine, pentostatin,cladribine or cytarabine) a corticosteroid (e.g., prednisone,prednisolone or methylprednisolone), an immunomodulatory agent (e.g.,lenalidomide), an antibiotic (e.g., doxorubicin, daunorubicin idarubicinor mitoxentrone), a synthetic flavon (e.g., flavopiridol), a Bcl2antagonist, (e.g., oblimersen or ABT-263), a hypomethylating agent(e.g., azacytidine or decitabine), an FLT3 inhibitor (e.g., midostaurin,sorafenib and AC220). Such molecules are suitably present in combinationin amounts that are effective for the purpose intended.

The antibodies of the present disclosure may be formulated in anysuitable form for delivery to a target cell/tissue,for example, asmicrocapsules or macroemulsions (Remington's Pharmaceutical Sciences,16th edition, Osol, A. Ed. (1980); Park et al., Molecules 10: 146-161(2005); Malik et al., Curr. Drug. Deliv. 4: 141-151 (2007)); assustained release formulations (Putney and Burke, Nature Biotechnol. 16:153-157, (1998)) or in liposomes (Maclean et al., Int. J. Oncol. 11:235-332 (1997); Kontermann, Curr. Opin. Mol. Ther. 8: 39-45 (2006)).

An antibody provided herein can also be entrapped in microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed, forexample, in Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa.

Various delivery systems are known and can be used to administer aprophylactic or therapeutic agent (e.g., an antibody that binds to GFRALas described herein), including, but not limited to, encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody, receptor-mediated endocytosis (see, e.g., Wuand Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleicacid as part of a retroviral or other vector, etc. In anotherembodiment, a prophylactic or therapeutic agent, or a compositionprovided herein can be delivered in a controlled release or sustainedrelease system. In one embodiment, a pump may be used to achievecontrolled or sustained release (see, e.g., Langer, supra; Sefton, 1987,CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In anotherembodiment, polymeric materials can be used to achieve controlled orsustained release of a prophylactic or therapeutic agent (e.g., anantibody that binds to GFRAL as described herein) or a composition ofthe invention (see, e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, 1983, J.,Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT PublicationNo. WO 99/20253). Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In oneembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable.

In yet another embodiment, a controlled or sustained release system canbe placed in proximity of the therapeutic target, for example, the nasalpassages or lungs, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Controlled release systems arediscussed, for example, by Langer (1990, Science 249:1527-1533). Anytechnique known to one of skill in the art can be used to producesustained release formulations comprising one or more antibodies thatbind to GFRAL as described herein. (See, e.g., U.S. Patent No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179- 189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro. Intl.Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997,“Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery,” Proc. Intl. Symp. Control Rel. Bioact. Mater.24:759-760).

Additional delivery systems can be used to administer a prophylactic ortherapeutic agent (e.g., an antibody that binds to GFRAL as describedherein) including, but not limited to, injectable drug delivery devices.Injectable drug delivery devices for anti-GFRAL antibodies include, forexample, hand-held devices or wearable devices. Hand-held devices usefulfor anti-GFRAL antibodies include autoinjectors, such as the FLEXIQ-DV(Elcam Medical) or PRO-JECT (Aptar Pharma) autoinjectors. Wearabledevices useful for anti-GFRAL antibodies include, for example, on-bodydrug delivery systems, such as the NEULASTA drug delivery kit (Amgen).Injectable drug delivery devices for anti-GFRAL antibodies can containthe antibody as a prophyactic or therapeutic agent, for example, inprefilled syringes, cartridges or vials. Injectable drug deliverydevices (e.g., autoinjectors) and/or their containers (e.g., syringes,cartridges or vials) for anti-GFRAL antibodies can be disposable.Exemplary injectable devices useful for anti-GFRAL antibodies aredescribed in WO2014/081780.

In some embodiments, additional drug delivery systems can be used toadminister a prophylactic or therapeutic agent (e.g., an antibody thatbinds to GFRAL as described herein) including, but not limited to,osmotic pumps. Different types of osmotic pump systems for anti-GFRALantibodies can be used, including, for example, single compartmentsystems, dual compartment systems, and multiple compartment systems.Exemplary osmotic pump systems useful for anti-GFRAL antibodies aredescribed, for example, in Herrlich et al. (2012) Advanced Drug DeliveryReviews 64, 1617-1627. In some embodiments, the osmotic pump foranti-GFRAL antibodies can include implantable drug-dispensing osmoticpumps such as the DUROS pump.

Therapeutic Methods

An anti-GFRAL antibody of the present disclosure may be used in, forexample, therapeutic methods.

In some embodiments, the present disclosure provides methods fortreating or preventing a GDF15-mediated disease, disorder, or condition,wherein the method comprises administering an anti-GFRAL antibody orfragment thereof described herein to a subject suffering from aGDF15-mediated disease, disorder or condition. Additionally, the subjectcan be administered a pharmaceutical composition comprising ananti-GFRAL antibody or fragment thereof described herein.

In some embodiments, present disclosure provides a method to treat asubject suffering from involuntary weight loss. An example of a suitablesubject may be one who is diagnosed with a wasting disease or cachexia.Suitable patients include those suffering from liver cirrhosis,hyperthyroidism, chronic kidney disease, Parkinson's disease, cancer,eating disorder (e.g., anorexia nervosa), chronic inflammatory disease(e.g., rheumatoid arthritis), sepsis or other forms of systemicinflammation, chronic obstructive pulmonary disease, AIDS, tuberculosis,and muscle wasting, such as muscular dystrophy or multiple sclerosis),or sarcopenia.

In some embodiments, the present disclosure also provides methods forpreventing involuntary weight loss in a subject who may be at risk ofinvoluntary weight loss due to a chronic disease, such as, livercirrhosis, hyperthyroidism, chronic kidney disease, Parkinson's disease,cancer, eating disorder (e.g., anorexia nervosa), chronic inflammatorydisease (e.g., rheumatoid arthritis), sepsis or other forms of systemicinflammation, chronic obstructive pulmonary disease, AIDS, tuberculosis,and muscle wasting, such as muscular dystrophy or multiple sclerosis),or sarcopenia. Such subjects may include subjects who have elevatedlevels of GDF15, are undergoing treatment for cancer, and the like.

In some embodiments, the present disclosure provides a method to treat asubject suffering from cachexia. An example of a suitable subject is onewho is diagnosed with cachexia. The present disclosure also providesmethods for preventing involuntary weight loss in a subject who may beat risk of involuntary weight loss due to onset of cachexia. Suchsubjects include subjects who have elevated levels of GDF15, havecancer, are undergoing treatment for cancer, have an eating disorder,and the like.

Also disclosed is a method for modulating GDF15 activity in a patienthaving elevated GDF15 activity. As used herein, “elevated GDF15activity” refers to increased activity or amount of GDF15 in abiological fluid of a subject in comparison to a normal subject. Anumber of conditions are associated with increased GDF15 serum level,wherein the increased GDF15 results in a number of symptoms such asappetite loss, weight loss, and the like. Examples of conditionsassociated with increased GDF15 serum level include cancer, e.g.,melanoma, gastric cancer, pancreatic cancer, prostate cancer; autoimmunediseases such as, arthritis and inflammation; cardiovascular diseaseslike atherosclerosis, heart failure, hypertension, myocardialinfarction, chest pain, and cardiovascular events; metabolic diseaseslike anemia, cachexia, anorexia, kidney disease, and thalassemia, etc.

A subject having any of the above diseases, disorders or conditions is asuitable candidate for receiving an anti-GFRAL antibody or fragmentthereof described herein, or a combination of a therapeutic agent andthe anti-GFRAL antibody or fragment thereof.

Administering the subject an anti-GFRAL antibody or fragment thereof tosuch a subject can decrease or prevent one or more of the symptomsassociated with a GDF15-mediated disease, disorder or condition. Forexample, administering an anti-GFRAL antibody or fragment thereof of thepresent disclosure can increase body weight and/or appetite in asubject. As another example, administering an anti-GFRAL antibody orfragment thereof of the present disclosure can maintain body weight in asubject or reduce body weight loss is a subject.

In one aspect, methods are provided for treating a disease, disorder orcondition comprising administering to an individual an effective amountof an anti-GFRAL antibody or fragment thereof. In certain embodiments, amethod for treating a disease, disorder, or condition comprisesadministering to an individual an effective amount of a pharmaceuticalformulation comprising an anti-GFRAL antibody and, optionally, at leastone additional therapeutic agent, such as those described herein.

An anti-GFRAL antibody or fragment thereof can be administered to ahuman for therapeutic purposes. Moreover, an anti-GFRAL antibody orfragment thereof can be administered to a non-human mammal expressingGFRAL with which the antibody cross-reacts (e.g., a primate, pig, rat,or mouse) for veterinary purposes or as an animal model of humandisease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the presentdisclosure (e.g., testing of dosages and time courses ofadministration).

Antibodies of the present disclosure can be used either alone or incombination with other compositions in a therapy. For example, ananti-GFRAL antibody of the present disclosure may be co-administeredwith at least one additional therapeutic agent and/or adjuvant. In someembodiments, the additional compound is a therapeutic antibody otherthan an anti-GFRAL antibody.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of an anti-GFRAL antibody or fragment thereof of thepresent disclosure can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.Antibodies of the present disclosure can also be used in combinationwith additional therapeutic regimens including, without limitation,those described herein.

An anti-GFRAL antibody of the present disclosure (and any additionaltherapeutic agent or adjuvant) can be administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody or conjugate issuitably administered by pulse infusion, particularly with decliningdoses of the antibody or fragment thereof. Dosing can be by any suitableroute, for example, by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

Antibodies of the present disclosure would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual subject, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theanti-GFRAL antibody need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of antibody or immunoconjugate present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of a disease, disorder, or condition,the appropriate dosage of an anti-GFRAL antibody or fragment thereof ofthe present disclosure (when used alone or in combination with one ormore other additional therapeutic agents, such as agents describedherein) will depend on the type of disease, disorder, or condition, tobe treated, the type of antibody, the severity and course of thedisease, disorder, or condition, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the subject'sclinical history and response to the antibody, and the discretion of theattending physician. The anti-GFRAL antibody or fragment thereof issuitably administered to the subject at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 100 mg/kg (e.g., 0.1mg/kg-20mg/kg, 1mg/kg-15mg/kg, etc.) ofantibody or fragment thereof can be an initial candidate dosage foradministration to the subject, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. Exemplary dosages of the antibody or fragment thereof may be inthe range from about 0.05 mg/kg to about 10.0 mg/kg. Thus, one or moredoses of about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg,5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, or 10.0 mg/kg (orany combination thereof) of antibody may be administered to the subject.Such doses may be administered intermittently, e.g., every week or everythree weeks (e.g., such that the subject receives from about two toabout twenty, or e.g., about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. An exemplary dosing regimen comprises administering aninitial loading dose, followed by a maintenance dose (e.g., weekly) ofthe antibody or fragment thereof. The initial loading dose may begreater than the maintenance dose. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

In some embodiments, the method described herein involves administeringthe subject an anti-GFRAL antibody or fragment thereof to a patient whohas involuntary body weight loss or is at risk of developing involuntarybody weight loss. The subject methods include administering ananti-GFRAL antibody or fragment thereof disclosed herein to a subjectwho has elevated serum levels of GDF15. In certain embodiment, theantibody or fragment thereof is an anti-GFRAL antibody that competeswith GDF15 for binding to extracellular domain of GFRAL. In certainembodiments, the antibody or fragment thereof binds to an extracellulardomain of a GFRAL protein but does not activate RET. For example, theantibody or fragment thereof is an anti-GFRAL antibody that competeswith GDF15 for binding to extracellular domain of GFRAL but does notactivate RET upon binding to GFRAL. Such an antibody is describedherein.

In the methods of the present disclosure, anti-GFRAL antibodies orfragments thereof described herein or pharmaceutical compositionscomprising said antibodies or fragments thereof can be administered to asubject (e.g., a human patient) to, for example, achieve a target bodyweight and/or maintain body weight; achieve a target body mass index(BMI) and/or maintain a BMI; increase appetite; and the like. A normalhuman adult has a BMI in the range 18.5-24.9 Kg/m². The subjecttreatment methods can increase body weight, BMI, muscle weight, and/orfood intake in a patient by at least about 5%, e.g., 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50% or more.

The methods relating to treatment or prevention of a GDF15-mediateddisease, disorder or condition (e.g., involuntary weight loss) describedherein include, for example, use of an anti-GFRAL antibody or fragmentthereof described herein for therapy/prevention alone or in combinationwith other types of therapy. The method involves administering to asubject the anti-GFRAL antibody or fragment thereof and another agent.

In some embodiments, the agent is administered to a patient experiencingloss of muscle mass, for example, loss of muscle mass associated with anunderlying disease. Underlying diseases associated with cachexiainclude, but are not limited to, cancer, chronic renal disease, chronicobstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatorydiseases, sepsis and other forms of systemic inflammation, musclewasting, such as muscular dystrophy, and the eating disorder known asanorexia nervosa. In some embodiments, the agent inhibits loss of leanmass (e.g., muscle mass) and or fat mass by at least 40%, 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100%.

In some embodiments, a loss of lean mass (e.g., muscle mass) isaccompanied by a loss of fat mass. In some embodiments, the agent caninhibit loss of fat mass by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%,98%, 99% or 100%.

In some embodiments, the agent is administered to a patient diagnosedwith body weight loss (e.g., involuntary weight loss). In someembodiments, the agent can revert body weight loss (e.g., involuntaryweight loss) by at least 2%, 5%, 10%, 15%, 20%, 25%, 30% or 35%.

In some embodiments, the agent is administered to a patient diagnosedwith loss of organ mass, for example, loss of organ mass associated withan underlying disease. Underlying diseases associated with cachexiainclude, but are not limited to, cancer, chronic renal disease, chronicobstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatorydiseases, sepsis and other forms of systemic inflammation, musclewasting, such as muscular dystrophy, and the eating disorder known asanorexia nervosa. In some embodiments, the agent can inhibit loss oforgan mass by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or100%. In some embodiments, loss of organ mass is observed in heart,liver, kidney, and/or spleen. In some embodiments, the loss of organmass in accompanied by a loss of muscle mass, a loss of fat mass and/orinvoluntary weight loss.

Sarcopenia, muscle wasting disorders and significant muscle weight losscan occur in the absence of cachexia, decreased appetite or body weightloss. In some embodiments, the agent can be used to treat a subjectdiagnosed with sarcopenia, a muscle wasting disorder and/or significantmuscle weight loss, whether or not the subject has, or has beendiagnosed with, cachexia or decreased appetite. Such a method comprisesadministering a therapeutically effective amount of one or more agentsto a subject in need thereof.

Any of a wide variety of therapies directed to treating or preventingcachexia can be combined in a composition or therapeutic method with thesubject proteins.

Where the GFRAL-ECD protein is administered in combination with one ormore other therapies, the combination can be administered anywhere fromsimultaneously to up to 5 hours or more, e.g., 10 hours, 15 hours, 20hours or more, prior to or after administration of a subject protein. Incertain embodiments, a subject protein and other therapeuticintervention are administered or applied sequentially, e.g., where asubject protein is administered before or after another therapeutictreatment. In yet other embodiments, a subject protein and other therapyare administered simultaneously, e.g., where a subject protein and asecond therapy are administered at the same time, e.g., when the secondtherapy is a drug it can be administered along with a subject protein astwo separate formulations or combined into a single composition that isadministered to the subject. Regardless of whether administeredsequentially or simultaneously, as illustrated above, the treatments areconsidered to be administered together or in combination for purposes ofthe present disclosure.

Cytokines that are implicated in cachexia include Activin A and IL-6.Increased activin levels have been associated with cancer-associatedcachexia and gonadal tumors. See, e.g., Marino et al. (2013) CYTOKINE &GROWTH FACTOR REV. 24:477-484. Activin A is a member of the TGF-betafamily, and is a ligand of the activin type 2 receptor, ActRIIB. See,e.g., Zhou et al. (2010) CELL 142:531-543. Circulating levels of IL-6have been shown to correlate with weight loss in cancer patients, aswell as with reduced survival. See, e.g., Fearon et al. (2012) CELLMETABOLISM 16: 153-166.

Accordingly, in some embodiments, one or more inhibitors of Activin-A orthe Activin-A receptor, ActRIIB, IL-6 or the IL-6 receptor (IL-6R), canbe administered in combination with (for example, administered at thesame time as, administered before, or administered after) an anti-GFRALantibody or fragment thereof described herein. Exemplary inhibitors ofActivin A or ActRIIB, include, for example, an anti-Activin-A antibodyor an antigen binding fragment thereof, an anti-ActRIIB antibody or anantigen binding fragment thereof, a small molecule inhibitor ofActivin-A, a small molecule inhibitor of ActRIIB, and a ‘decoy’ receptorof ActRIIB, such as a soluble ActRIIB receptor and a fusion of thesoluble ActRIIB receptor with an Fc molecule (ActRIIB-Fc). See, e.g.,Zhou et al. (2010), supra. Suitable inhibitors of IL-6 or IL-6R, includean anti-IL-6 antibody or an antigen binding fragment thereof, ananti-IL-6R antibody or an antigen binding fragment thereof, a smallmolecule inhibitor of IL-6, a small molecule inhibitor of IL-6R, and a‘decoy’ receptor of IL-6R, such as a soluble IL-6 receptor and a fusionof the soluble IL-6 receptor with an Fc molecule (IL6R-Fc). See, e.g.,Enomoto et al. (2004) BIOCHEM. AND BIOPHYS. RES. COMM. 323: 1096-1 102;Argiles et al. (2011) EUR. J. PHARMACOL. 668:S81-S86; Tuca et al. (2013)ONCOLOGY/HEMATOLOGY 88:625-636. Suitable inhibitors of IL-6 or IL-6R caninclude, e.g., Tocilizumab (Actemra®, Hoffmann-LaRoche), a humanizedanti-IL-6R monoclonal antibody approved for treatment of rheumatoidarthritis, and Sarilumab/REGN88 (Regeneron), a humanized anti-IL6Rantibody in clinical development for treatment of rheumatoid arthritis;and Selumetinib/AZD6244 (AstraZeneca), an allosteric inhibitor of MEK,which has been shown to inhibit IL-6 production. Prado et al. (2012)BRITISH J. CANCER 106: 1583-1586.

TNFα and IL-1 are cytokines known to be involved in mediation of theproinflammatory response, which are also implicated in muscle depletion,anorexia and cachexia. Increased circulating levels of TNFα appear toinhibit myogenesis. TNFα, also known as “cachectin,” stimulatesinterleukin-1 secretion and is implicated in the induction of cachexia.IL-1 is a potent trigger of the acute-phase inflammatory response, andit has been shown that infusion of IL-1 can lead to marked weight lossand appetite loss. IL-1 has been shown to contribute to the initiationof cancer cachexia in mice bearing a murine colon-26 adenocarcinoma(Strassmann et al. (1993) J. IMMUNOL. 150:2341). See also, Mathys andBilliau (1997) NUTRITION 13 :763-770; Fong et al. (1989) AM. J.PHYSIOL. - REGULATORY, INTEGRATIVE AND COMPARATIVE PHYSIOL.,256:R659-R665. Thus, TNFα inhibitors and IL-1 inhibitors that are usedin the treatment of rheumatoid arthritis may also be useful in thetreatment of cachexia.

Accordingly, in some embodiments, one or more inhibitors of TNFα or IL-1can be administered in combination with (e.g., administered at the sametime as, administered before, or administered after) an anti-GFRALantibody or fragment thereof described herein. Suitable inhibitors ofTNFα or IL-1 include an anti-TNFα antibody or an antigen bindingfragment thereof, an anti-IL- 1 antibody or an antigen binding fragmentthereof, a small molecule inhibitor of TNFα or IL-1, and a ‘decoy’receptor of TNFα or IL-1, such as a soluble TNFα or IL-1 receptor and afusion of the soluble form of TNFα or IL-1 with an Fc molecule. Suitableinhibitors of TNFα include for example, etanercept (Enbrel®,Pfizer/Amgen), infliximab (Remicade®, Janssen Biotech), adalimumab(Humira®, Abbvie), golimumab (Simponi®, Johnson and Johnson/Merck), andcertolizumab pegol (Cimzia®, UCB). Suitable IL-1 inhibitors include,e.g., Xilonix® antibody that targets IL-1 a (XBiotech), anikinra(Kinaret®, Amgen), canakinumab (Ilaris® , Novartis), and rilonacept(Arcalyst®, Regeneron). In certain embodiments, the TNFα inhibitor orIL-1 inhibitor, which is typically administered systemically for thetreatment of rheumatoid arthritis may be administered locally anddirectly to the tumor site.

Myostatin, also known as GDF-8, is a member of the TGF-β family ofpeptides that is a negative regulator of muscle mass, as shown byincreased muscle mass in myostatin deficient mammals. Myostatin is aligand of the activin type 2 receptor, ActRIIB.

Accordingly, in some embodiments, one or more inhibitors of myostatin orits receptor may be administered in combination with (for example,administered at the same time as, administered before, or administeredafter) an anti-GFRAL antibody or fragment thereof described herein.Suitable inhibitors of myostatin or ActRIIB, include an anti-myostatinantibody or an antigen binding fragment thereof, an anti-ActRIIBantibody or an antigen binding fragment thereof, a small moleculeinhibitor of myostatin, a small molecule inhibitor of ActRIIB, and a‘decoy’ receptor of GDF-8, such as a soluble ActRIIB and a fusion of thesoluble form of ActRIIB with an Fc molecule. See, e.g., Lokireddy et al.(2012) BIOCHEM. J. 446(I):23-26. Myostatin inhibitors that may besuitable for the present methods include REGN1033 (Regeneron); seeBauerlein et al. (2013) J. CACHEXIA SARCOPENIA MUSCLE: Abstracts of the7th Cachexia Conference, Kobe/Osaka, Japan, Dec. 9-11, 2013, Abstract4-06; LY2495655 (Lilly), a humanized anti-myostatin antibody in clinicaldevelopment by Eli Lilly; see also “A PHASE 2 STUDY OF LY2495655 INPARTICIPANTS WITH PANCREATIC CANCER,” available on the world wide web atclinicaltrials.gov/ct2/NCT01505530; NML identifier: NCT01505530; ACE-031(Acceleron Pharma); and stamulumab (Pfizer).

Agents such as Ghrelin or ghrelin mimetics, or other growth hormonesecretagogues (GHS) which are able to activate the GHS receptor(GHS-RIa), also known as the ghrelin receptor, can be useful forincreasing food intake and body weight in humans. See Guillory et al.(2013) in VITAMINS AND HORMONES vol. 92, chap.3; and Steinman and DeBoer(2013) VITAMINS AND HORMONES vol. 92, chap. 8.

Accordingly, in some embodiments, one or more Ghrelin or ghrelinmimetics, or other growth hormone secretagogues (GHS), can beadministered in combination with (for example, administered at the sametime as, administered before, or administered after) an anti-GFRALantibody or fragment thereof described herein. Suitable ghrelin mimeticsinclude anamorelin (Helsinn, Lugano, CH); see Temel et al. (2013) J.CACHEXIA SARCOPENIA MUSCLE: Abstracts of the 7th Cachexia Conference,Kobe/Osaka, Japan, Dec. 9-11, 2013, Abstract 5-01. Other suitable GHSmolecules can be identified, for example, using the growth hormonesecretagogue receptor Ghrelin competition assay described in PCTPublication Nos. WO2011/117254 and WO2012/113103.

Agonists of the androgen receptor, including small molecules and otherselective androgen receptor modulators (SARMs) can be useful in treatingcachexia and/or sarcopenia. See, e.g., Mohler et al. (2009) J. MED.CHEM. 52:3597-3617; Nagata et al. (2011) BIOORGANIC AND MED. CHEM.LETTERS 21 : 1744-1747; and Chen et al. (2005) MOL. INTERV. 5: 173-188.Ideally, SARMs should act as full agonists, like testosterone, inanabolic target tissues, such as muscle and bone, but should demonstrateonly partial or pure androgen receptor antagonistic activities onprostate tissue. See, e.g., Bovee et al. (2010) J. STEROID BIOCHEM. &MOL. BIOL. 118:85-92. Suitable SARMs can be identified, e.g., by use ofthe methods and assays described in Zhang et al. (2006) BIOORG. MED.CHEM. LETT. 16:5763-5766; and Zhang et al. (2007) BIOORG. MED. CHEM.LETT. 17:439-443.

Accordingly, in some embodiments, one or more androgen receptor agonistscan be administered in combination with (for example, administered atthe same time as, administered before, or administered after) ananti-GFRAL antibody or fragment thereof described herein. Suitable SARMsinclude, for example, GTx-024 (enobosarm, Ostarine®, GTx, Inc.), a SARMin phase II clinical development by GTx, Inc. See also, Dalton et al.(2011) J. CACHEXIA SARCOPENIA MUSCLE 2: 153-161. Other suitable SARMsinclude 2-(2,2,2)-trifluoroethyl-benzimidazoles (Ng et al. (2007)BIOORG. MED. CHEM. LETT. 17: 1784-1787) and JNJ-26146900 (Allan et al.(2007) J. STEROID BIOCHEM. & MOL. BIOL. 103:76-83).

β-adrenergic receptor blockers, or beta-blockers, have been studied fortheir effect on body weight in cachexia subjects, and have beenassociated with partial reversal of cachexia in patients with congestiveheart failure. See, e.g., Hryniewicz et al. (2003) J. CARDIAC FAILURE9:464-468. Beta-blocker MT-102 (PsiOxus Therapeutics, Ltd.) has beenevaluated in a phase 2 clinical trial for subjects with cancer cachexia.See Coats et al. (2011) J. CACHEXIA SARCOPENIA MUSCLE 2:201-207.Accordingly, in some embodiments, one or more β-adrenergic receptorblockers, or beta-blockers, can be administered in combination with (forexample, administered at the same time as, administered before, oradministered after) an anti-GFRAL antibody or fragment thereof describedherein.

Melanocortin receptor-knockout mice with a genetic defect inmelanocortin signaling exhibit a phenotype opposite that of cachexia:increased appetite, increased lean body mass, and decreased metabolism.Thus, melanocortin antagonism has emerged as a potential treatment forcachexia associated with chronic disease (DeBoer and Marks (2006) TRENDSIN ENDOCRINOLOGY AND METABOLISM 17: 199-204).

Accordingly, in some embodiments, one or more inhibitors of amelanocortin peptide or a melanocortin receptor can be administered incombination (e.g., administered at the same time as, administeredbefore, or administered after) with an anti-GFRAL antibody or fragmentthereof described herein. Suitable inhibitors of melanocortins ormelanocortin receptors include an anti-melanocortin peptide antibody oran antigen binding fragment thereof, an anti-melanocortin receptorantibody or an antigen binding fragment thereof, a small moleculeinhibitor of a melanocortin peptide, a small molecule inhibitor of amelanocortin receptor, and a ‘decoy’ receptor of a melanocortinreceptor, such as soluble melanocortin receptor and a fusion of asoluble melanocortin receptor with an Fc molecule. Suitable melacortinreceptor inhibitors include, for example, the melanocortin receptorantagonist agouri-related peptide (AgRP(83-132)), which has beendemonstrated to prevent cachexia-related symptoms in a mouse model ofcancer-related cachexia (Joppa et al. (2007) PEPTIDES 28:636-642).

Anti-cancer agents, especially those that can cause cachexia and elevateGDF15 levels, such as cisplatin, can be used in methods of the presentdisclosure in combination with (for example, administered at the sametime as, administered before, or administered after) an anti-GFRALantibody or fragment thereof described herein. Many cancer patients areweakened by harsh courses of radio- and/or chemotherapy, which can limitthe ability of the patient to tolerate such therapies, and hencerestrict the dosage regimen. Certain cancer agents themselves, such asfluorouracil, adriamycin, methotrexate and cisplatin, can contribute tocachexia, for example by inducing severe gastrointestinal complications.See, e.g., Inui (2002) CANCER J. FOR CLINICIANS 52:72-91. By the methodsof the present disclosure, in which an anti-cancer agent is administeredin combination with an anti-GFRAL antibody of the disclosure, it ispossible to decrease the incidence and/or severity of cachexia, andultimately increase the maximum tolerated dose of such an anti-canceragent. Accordingly, efficacy of treatment with anti-cancer agents thatcan cause cachexia can be improved by reducing the incidence of cachexiaas a dose-limiting adverse effect, and by allowing administration ofhigher doses of a given anticancer agent.

Thus, provided herein are pharmaceutical compositions comprising ananti-GFRAL antibody or fragment thereof described herein in combinationwith an agent selected from the group consisting of an inhibitor ofActivin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitorof IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, aTNFα inhibitor, an IL-Iα inhibitor, a myostatin inhibitor, abeta-blocker, a melanocortin peptide inhibitor, a melanocortin receptorinhibitor, and an anti-cancer agent. The present disclosure alsoincludes methods of treating, preventing or minimizing cachexia and/orsarcopenia in a mammal comprising administering to a mammal in needthereof a pharmaceutical composition or compositions comprising aneffective amount of an anti-GFRAL antibody of the disclosure incombination with an effective amount of an inhibitor of Activin-A, aninhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, aghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFαinhibitor, an IL-Iα inhibitor, a myostatin inhibitor, a beta-blocker, amelanocortin peptide inhibitor, or a melanocortin receptor inhibitor.

In another aspect, provided herein is a method of inhibiting loss ofmuscle mass associated with an underlying disease comprisingadministering to a mammal in need thereof a pharmaceutical compositionor compositions comprising an effective amount of an anti-GFRAL antibodyor fragment thereof described herein in combination with an effectiveamount of an inhibitor of Activin-A, an inhibitor of ActRIIB, aninhibitor of IL-6 or an inhibitor of IL-6R, a ghrelin, a ghrelin mimeticor a GHS-RIa agonist, a SARM, a TNFα inhibitor, an IL-Iα inhibitor, amyostatin inhibitor, a beta-blocker, a melanocortin peptide inhibitor,or a melanocortin receptor inhibitor to prevent or reduce loss of musclemass. The underlying disease can be selected from the group consistingof cancer, chronic heart failure, chronic kidney disease, chronicobstructive pulminary disease, AIDS, multiple sclerosis, rheumatoidarthritis, sepsis, and tuberculosis. Additionally, in some embodiments,the loss of muscle mass is accompanied by a loss of fat mass.

In another aspect, provided herein is a method of inhibiting or reducinginvoluntary weight loss in a mammal comprising administering to a mammalin need thereof a pharmaceutical composition or pharmaceuticalcompositions comprising an effective amount of an anti-GFRAL antibody ofthe disclosure in combination with an effective amount of an inhibitorof Activin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or aninhibitor of IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, aSARM, a TNFα inhibitor, a IL-Iα inhibitor, a myostatin inhibitor, abeta-blocker, a melanocortin peptide inhibitor, or a melanocortinreceptor inhibitor.

Certain anti-cancer agents, such as cisplatin, have one or moreundesirable adverse effects that involve causing or increasing one ormore syndromes such as cachexia, sarcopenia, muscle wasting, bonewasting or involuntary body weight loss. Accordingly, in another aspect,provided herein is a method of treating cancer, while preventing,minimizing or reducing the occurrence, frequency or severity ofcachexia, sarcopenia, or muscle wasting, bone wasting or involuntaryloss of body weight in a mammal, comprising administering to a mammal inneed thereof a pharmaceutical composition comprising an effective amountof an anti-GFRAL antibody or fragment thereof described herein incombination with one or more anti-cancer agents. In some embodiments,the method of treating cancer, while preventing, minimizing or reducingthe occurrence, frequency or severity of cachexia, sarcopenia or musclewasting, bone wasting or involuntary loss of body weight in a mammal,comprises administering to a mammal in need thereof a pharmaceuticalcomposition comprising an effective amount of an anti-GFRAL antibody orfragment thereof described herein in combination with one or moreanti-cancer agents known to cause or increase the occurrence, frequencyor severity of cachexia, sarcopenia, or muscle wasting, bone wasting orinvoluntary loss of body weight in a mammal.

Diagostic Methods and Methods of Detection

In one aspect, anti-GFRAL antibodies and fragments thereof of thepresent disclosure are useful for detecting the presence of GFRAL in abiological sample. Such anti-GFRAL antibodies can include those thatbind to human GFRAL. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue.

In one aspect, the present disclosure provides a method of detecting thepresence of GFRAL in a biological sample. In certain embodiments, themethod comprises contacting the biological sample with an anti-GFRALantibody under conditions permissive for binding of the anti-GFRALantibody to GFRAL, and detecting whether a complex is formed between theanti-GFRAL antibody and GFRAL.

In one aspect, the present disclosure provides a method of diagnosing adisorder associated with expression of GFRAL. In certain embodiments,the method comprises contacting a test cell with an anti-GFRAL antibody;determining the level of expression (either quantitatively orqualitatively) of GFRAL by the test cell by detecting binding of theanti-GFRAL antibody to GFRAL; and comparing the level of expression ofGFRAL by the test cell with the level of expression of GFRAL by acontrol cell (e.g., a normal cell of the same tissue origin as the testcell or a cell that expresses GFRAL at levels comparable to such anormal cell), wherein a higher level of expression of GFRAL by the testcell as compared to the control cell indicates the presence of adisorder associated with increased expression of GFRAL. In certainembodiments, the test cell is obtained from an individual suspected ofhaving a disease, disorder or condition associated with expression ofGDF15 and/or a disease, disorder or condition in which it is desirableto inhibit the in vivo effects of GDF15. In certain embodiments, thedisease, disorder or condition is, for example, involuntary weight loss.Such exemplary diseases, disorders or conditions may be diagnosed usingan anti-GFRAL antibody of the present disclosure.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-GFRALantibody to GFRAL expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing GFRAL on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-GFRAL antibody under conditions permissive for binding of theanti-GFRAL antibody to GFRAL, and detecting whether a complex is formedbetween the anti-GFRAL antibody and GFRAL on the cell surface. Anexemplary assay for detecting binding of an anti-GFRAL antibody to GFRALexpressed GFRAL on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-GFRALantibodies to GFRAL. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-GFRAL antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, for example, throughan enzymatic reaction or molecular interaction. Exemplary labelsinclude, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H,and ¹³¹I, fluorophores such as rare earth chelates or fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, for example, firefly luciferase and bacterial luciferase(see, e.g., U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

In certain embodiments, anti-GFRAL antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-GFRALantibody from any GFRAL that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-GFRALantibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (see, e.g., Bennich et al., U.S. Pat.No. 3,720,760), or by covalent coupling (for example, usingglutaraldehyde cross-linking), or by insolubilizing the anti-GFRALantibody after formation of a complex between the anti- GFRAL antibodyand GFRAL, for example, by immunoprecipitation.

Any of the above embodiments of diagnosis or detection may be carriedout using an immunoconjugate of the present disclosure in place of or inaddition to an anti-GFRAL antibody.

Assays

Anti-GFRAL antibodies of the present disclosure may be characterized fortheir physical/chemical properties and/or biological activities byvarious assays known in the art.

1. Activity Assays

In one aspect, assays are provided for identifying anti-GFRAL antibodiesthereof having biological activity. Biological activity can include, forexample, assays which measure effects on glucose and/or lipidmetabolism. For example, a blood glucose assay can be used. Bloodglucose (e.g., in mouse tail snip or in a human blood sample) can bemeasured using ACCU-CHEK Active test strips read by ACCU-CHEK Activemeter (Roche Diagnostics, Indianapolis, Ind.) following manufacturer'sinstruction. In addition, for example, a lipid profile assay can beused. Whole blood (e.g., from mouse tail snips or from a human bloodsample) can be collected into plain capillary tubes (BD Clay AdamsSurePrep, Becton Dickenson and Co. Sparks, Md.). Serum and blood cellscan be separated by spinning the tubes in an Autocrit Ultra 3 (BectonDickinson and Co. Sparks, Md.). Serum samples can be assayed for lipidprofile (triglyceride, total cholesterol, HDL, and non-HDL) usingIntegra 400 Clinical Analyzer (Roche Diagnostics, Indianapolis, Ind.)following the manufacturer's instructions.

2. Binding Assays and Other Assays

In one aspect, an anti-GFRAL antibody is tested for its antigen bindingactivity. For example, in certain embodiments, an anti-GFRAL antibody istested for its ability to bind to exogenous or endogenous GFRALexpressed on the surface of a cell. A FACS assay may be used for suchtesting.

A panel of monoclonal antibodies raised against GFRAL may be groupedbased upon the epitiopes they recognize, a process known as epitopebinning. Epitope binning is typically carried out using competitionassays, which evaluate an antibody's ability to bind to an antigen inthe presence of another antibody. In an exemplary competition assay,immobilized GFRAL is incubated in a solution comprising a first labeledantibody that binds to GFRAL and a second unlabeled antibody that isbeing tested for its ability to compete with the first antibody forbinding to GFRAL. The second antibody may be present in a hybridomasupernatant. As a control, immobilized GFRAL is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to GFRAL, excess unbound antibody is removed, and theamount of label associated with immobilized GFRAL is measured. If theamount of label associated with immobilized GFRAL is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to GFRAL. In certain embodiments, immobilized GFRAL ispresent on the surface of a cell or in a membrane preparation obtainedfrom a cell expressing GFRAL on its surface.

High-throughput methods of epitope binning are also known in the art(see, e.g., Jia et al., J. Immunol. Methods 2004, 288(1-2):91-98,describing a method of multiplexed competitive antibody binning for thecharacterization of monoclonal antibodies; and Miller et al., J.Immunol. Methods 2011, 365(1-2):118-25, describing epitope binning ofmurine monoclonal antibodies by a multiplexed pairing assay).

3. Epitope Mapping

Epitope mapping is the process of identifying the binding sites, orepitopes, of an antibody on its target protein antigen (e.g., epitopesof an anti-GFRAL antibody on GFRAL). Antibody epitopes may be linearepitopes or conformational epitopes. Linear epitopes are formed by acontinuous sequence of amino acids in a protein. Conformational epitopesare formed of amino acids that are discontinuous in the proteinsequence, but which are brought together upon folding of the proteininto its three-dimensional structure.

A variety of methods are known in the art for mapping antibody epitopeson target protein antigens. These include mutagenesis methods, peptidescanning methods, display methods, methods involving and massspectroscopy, and structural determination.

The site directed mutagenesis method involves targeted site-directedmutagenesis where critical amino acids are identified by systematicallyintroducing substitutions along the protein sequence and thendetermining the effects of each substitution on antibody binding. Thismay be done by “alanine scanning mutagenesis,” as described, forexample, by Cunningham and Wells (1989) Science 244: 1081-1085, or someother form of point mutagenesis of amino acid residues in human GFRAL.Mutagenesis studies, however, may also reveal amino acid residues thatare crucial to the overall three-dimensional structure of GFRAL but thatare not directly involved in antibody-antigen contacts, and thus othermethods may be necessary to confirm a functional epitope determinedusing this method.

Shotgun mutagenesis mapping utilizes a comprehensive plasmid-mutationlibrary for the target gene, with each clone in the library bearing aunique amino acid mutation and the entire library covering every aminoacid in the target protein. The clones that constitute the mutationlibrary are individually arranged in microplates, expressed withinliving mammalian cells, and tested for immunoreactivity with antibodiesof interest. Amino acids critical for antibody epitopes are identifiedby a loss of reactivity and are then mapped onto a protein structure tovisualize epitopes. By automating the analysis, new epitope maps can bederived within days to weeks. Because it uses the native structure ofproteins within mammalian cells, the technique allows both linear andconformational epitope structures to be mapped on complex proteins.(See, e.g., Paes et al., J. Am. Chem. Soc. 131(20): 6952-6954 (2009);Banik and Doranz, Genetic Engineering and Biotechnology News 3(2): 25-28(2010)).

The epitope bound by an anti-GFRAL antibody may also be determined usingpeptide scanning methods. In peptide scanning, libraries of shortpeptide sequences from overlapping segments of the target protein,GFRAL, are tested for their ability to bind antibodies of interest. Thepeptides are synthesized and screened for binding, e.g., using ELISA orBIACORE, or on a chip, by any of the multiple methods for solid-phasescreening (see, e.g., Reineke et al., Curr. Opin. Biotechnol. 12: 59-64,2001) as in the “pepscan” methodology (see, e.g., WO 84/03564; WO93/09872). Such peptide screening methods may not be capable ofdetecting some discontinuous functional epitopes, i.e. functionalepitopes that involve amino acid residues that are not contiguous alongthe primary sequence of the GFRAL polypeptide chain.

A recently developed technology termed CLIPS (chemical linkage ofpeptides onto scaffolds) may be used to map conformational epitopes. Theloose ends of the peptides are affixed onto synthetic scaffolds, so thatthe scaffolded peptide may be able to adopt the same spatial structureas the corresponding sequence in the intact protein. CLIPS technology isused to fix linear peptides into cyclic structures ('single-loop'format), and to bring together different parts of a protein binding site('double-loop', ‘triple-loop’, etc. format), so as to createconformational epitopes that may be assayed for antibody binding (see,e.g., U.S. Pat. No. 7,972,993).

The epitopes bound by anti-GFRAL antibodies of the present disclosuremay also be mapped using display techniques, including, for example,phage display, microbial display, and ribosome/mRNA display as describedabove. In these methods, libraries of peptide fragments are displayed onthe surface of the phage or cell. Epitopes are then mapped by screeningantibodies against these fragments using selective binding assays. Anumber of computational tools have been developed which allow theprediction of conformational epitopes based upon linearaffinity-selected peptides obtained using phage display (see,e.g.,Mayrose et al., Bioinformatics 23: 3244-3246 , 2007). Methods arealso available for the detection of conformational epitopes by phagedisplay. Microbial display systems may also be used to express properlyfolded antigenic fragments on the cell surface for identification ofconformational epitopes (see, e.g., Cochran et al., J. Immunol. Meth.287: 147-158, 2004; Rockberg et al., Nature Methods 5: 1039-1045, 2008).

Methods involving proteolysis and mass spectroscopy may also be used todetermine antibody epitopes (see, e.g., Baerga-Ortiz et al., ProteinSci. 2002 June; 11(6): 1300-1308). In limited proteolysis, the antigenis cleaved by different proteases, in the presence and in the absence ofthe antibody, and the fragments are identified by mass spectrometry. Theepitope is the region of the antigen that becomes protected fromproteolysis upon binding of the antibody (see, e.g., Suckau et al.,Proc. Natl. Acad. Sci. USA 87:9848-9852, 1990). Additional proteolysisbased methods include, for example, selective chemical modification(see, e.g., Fiedler et al., Bioconjugate Chemistry 1998, 9(2): 236-234,1998), epitope excision (see, e.g., Van de Water et al., Clin. Immunol.Immunopathol. 1997, 85(3): 229-235, 1997), and the recently developedmethod of hydrogen-deuterium (H/D) exchange (see, e.g., Flanagan, N.,Genetic Engineering and Biotechnology News 3(2): 25-28, 2010).

The epitope bound by anti-GFRAL antibodies of the present disclosure mayalso be determined by structural methods, such as X-ray crystalstructure determination (see, e.g., WO 2005/044853), molecular modelingand nuclear magnetic resonance (NMR) spectroscopy, including NMRdetermination of the H-D exchange rates of labile amide hydrogens whenfree and when bound in a complex with an antibody of interest (see,e.g., Zinn-Justin et al. (1992) Biochemistry 31:11335-11347; Zinn-Justinet al. (1993) Biochemistry 32:6884-6891).

Additional antibodies binding to the same epitope as an anti-GFRALantibody of the present disclosure may be obtained, for example, byscreening of antibodies raised against GFRAL o for binding to theepitope, by immunization of an animal with a peptide comprising afragment of human GFRAL comprising the epitope sequence, or by selectionof antibodies using phage display for binding to the epitope sequence.Antibodies that bind to the same functional epitope might be expected toexhibit similar biological activities, such as blocking a biologicalactivity of GFRAL, and such activities can be confirmed by functionalassays of the antibodies.

4. Additional Activity Assays

In one embodiment, an anti-GFRAL antibody of the present disclosure isan antagonist antibody that inhibits a biological activity of GFRAL. Theanti-GFRAL antibodies of the present disclosure may be assayed todetermine if they inhibit a biological activity of GFRAL.

In one aspect, purified anti-GFRAL antibodies can be furthercharacterized by a series of assays including, but not limited to,N-terminal sequencing, amino acid analysis, non-denaturing sizeexclusion high pressure liquid chromatography (HPLC), mass spectrometry,ion exchange chromatography and papain digestion.

In one embodiment, the present disclosure contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desirable candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the antibody are measured to ensurethat only the desired properties are maintained. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.An in vitro assay to assess ADCC activity of a molecule of interest isdescribed, for example, in U.S. Pat. Nos. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in a animal model such as that disclosed in Clyneset al. PNAS (USA) 95:652-656 (1998). C1q binding assays may also becarried out to confirm that the antibody is unable to bind C1q and hencelacks CDC activity. To assess complement activation, a CDC assay, forexample, as described in Gazzano-Santoro et al., J. Immunol. Methods202:163 (1996), may be performed. FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art.

Although the foregoing present disclosure has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the present disclosure. The disclosures of allpatent and scientific literatures cited herein are expresslyincorporated in their entirety by reference.

EXAMPLES

The following are examples of methods and compositions of the presentdisclosure.

Example 1: Generation of Antibodies

Antibodies to a GFRALprotein were generated, for example, byimmunizations of mice with cells expressing a GFRAL protein,co-expressing a RET protein and a GFRAL protein, or cross-linking of aGDF15 protein onto cells co-expressing a RETprotein and a GFRAL protein.Mice were also immunized with a GFRAL ECD, GDF15:GFRAL ECD complexand/or GFRAL ECD Fc fusion: RET ECD Fc fusion.

For example, the cells used for immunizations were prepared as follows.293EXPI (Invitrogen) cells were transiently transfected with nucleicacid sequences encoding a GFRAL protein (SEQ ID NO: 1797) orco-transfected with nucleic acid sequences encoding a GFRAL protein anda RET protein (SEQ ID NOS: 1797 and 1813). Cells were analyzed forexpression of GFRAL and RET by the respective specific antibodies byFACS. Cells were washed 2 times in PBS, pelleted by centrifugation andmembrane preps were generated. 129/B6 or NZBW animals were immunizedwith membrane preps with adjuvants. Animals were boosted to induce asuitable titer. Titers were determined by ELISA and/or FACS. Single cellsuspensions of lymphocytes were obtained from spleen and draining lymphnodes of animals with suitable titers. Cells were fused with SP2/0myeloma cells at a ratio of 1:12 by electrofusion. Fused cells wereplated into plates in the presence of HAT selection. After 10-14 days ofculture, supernatants were collected and subjected to screening by cellimaging by CellnSight using GFRAL or GFRAL and REToverexpressing-293EXPI cells or by ELISA using GFRAL-Fc protein or RETand GFRAL Fc-heterodimers to confirm binding. Positive clones werefurther selected and subjected to subcloning.

In multiple campaigns of immunizations and fusions, over one-hundredthousand hybridoma clones were screened and more than two thousandclones were selected for GDF15-binding, cell-based GDF15-inducedsignaling and cell-based GDF15-independent signaling. Hundreds of clones(e.g., 250) were selected for additional study, including assays forbinding affinity, domain mapping, and epitope specificity. Thousands ofhybridoma supernatants were also tested in functional assays, includingantagonistic and agonist activity assays. Hundreds of clones werepurified for further testing.

Example 2: Screening and Selection of Antibodies

After 2 weeks of culture, hybridoma supernatants were screened formonoclonal antibodies binding to a GFRAL protein by cell imaging byCelInSight using GFRAL or GFRAL and RET overexpressing-293EXPI cells orby ELISA using GFRAL-Fc protein or RET and GFRAL Fc-heterodimers.Briefly, screening by cell imaging, 293EXPI cells were transientlytransfected with nucleic acid sequences encoding GFRAL or co-transfectedwith nucleic acid sequences encoding GFRAL and RET. Transfected cellswere plated onto 384-well plates with clear bottom. Media was replaced24 hours post transfection. At 48 hrs, media was aspirated off theplates, hybridoma supernatants were added to the wells and incubated atroom temperature for 30 mins. Then A647 anti-mouse Fc were added to thewells and incubated at room temperature for another 30 mins. Dapipositive cells were analyzed for a signal in the A647 channel and apositive A647 signal indicates GFRAL binders.

Briefly, screening by ELISA, GFRAL-Fc protein or RET and GFRALFc-heterodimers was captured by anti-human Fc reagents coated onto ELISAplates. Plates were blocked using PBS/1% BSA. 15 μL of hybridomasupernatants were added to the wells and incubated at room temperaturefor 1 hr. After 3 washes, 15 μL of HRP-anti-mouse Fc secondary wereadded to the wells and incubated at room temperature for 1 hr. After 3washes, 15 μL of TMB were used to develop the plates. A positive signalindicates GFRAL binders.

From these assays, thousands of antibodies were identified as binders toGFRAL. These antibodies were subjected to further testing, whichincluded assaying for binding affinity, domain mapping, epitopespecificity, and agonistic and antagonistic function. Hundreds ofantibodies were purified for further testing.

In addition, the binding affinity of antibodies to human and mouse GFRALwere measured. For example, antibodies were rank ordered based on theirbinding affinity to human GFRAL and mouse GFRAL by low resolution K_(D)measurement by Biacore. Briefly, an anti-mouse Fc antibody(Sigma-Aldrich, St. Louis, Mo.) was immobilized on all four flow cellsof a CM5 chip using amine coupling reagents (GE Healthcare LifeSciences,Piscataway, N.J.). Purified antibodies were captured (˜100 RUs) on flowcells 2, 3 and 4 using flow cell 1 as a reference. This was followed byinjection of human or mouse GFRAL (25 nM in PBS-P buffer) at a flow rateof 70 μL/min and monitoring the binding kinetics at 25° C.

Binding affinity measurements were also made in additional Biacore basedassays. For example, equilibrium dissociation constant (K_(D))measurements were carried out with purified antibodies to evaluate theirbinding to human GFRAL or mouse GFRAL. As mentioned above, anti-mouse Fcantibody (Sigma-Aldrich, St. Louis, Mo.) was immobilized on all fourflow cells of a CM5 chip using amine coupling reagents (GE HealthcareLifeSciences, Piscataway, N.J.). Purified antibodies were captured (˜100RUs) on flow cells 2, 3 and 4 using flow cell 1 as a reference. This wasfollowed by injection of human or mouse GFRAL in PBS-P buffer) at a flowrate of 70 μL/min and the binding kinetics were evaluated at 25° C.

Representative results for binding affinity (e.g., K_(D) (nM)) to humanand mouse GFRAL are shown in Table 26 below. In addition, representativeresults for off-rate of binding (e.g., K_(off) (1/s)) are shown in Table26 below for exemplary antibodies.

TABLE 26 Binding Affinity Human GFRAL Mouse GFRAL Clone ID K_(D) (nM)k_(off) (1/s) K_(D) (nM) k_(off) (1/s) 1C1 <0.1  ~1E5 3.68  2.5E−3 25M22<0.1  ~1E−5 4.2   1E−4 8D8 0.46 1.24E−4 8.17 2.81E−3 12A3 <0.1  ~1E−55.7 2.16E−3 3P10 <0.1  ~1E−5 <0.1  ~1E−5 5F12 <0.1  ~1E−5 2.2   9E−45A20 0.081 3.32E−5 <0.1  4−E4 17J16 <0.1  ~1E−5 <0.1   1E−5 6G9 <0.1 ~1E5 No N/A 2B8 0.73  ~1E5 3.76  1.3E−2 6N16 0.16 9.93E−5 ND NA 8C10<0.1  7.2E−5 weak 49.26 2B11 0.1 8.45E−5 3.7 1.44E−3 1B3 <0.1  9.4E−94.4 1.07E−3 19K19 0.21 1.35E−4 4.7  3.4E−3 22N5 0.21 1.32E−4 3.479.43E−4 2A9 0.25 1.53E−4 3.6  2.9E−3 24G2 1.4 6.96E−4 5.02 1.73E−3 2I230.23 6.77E−5 ND NA 1A3 0.11 7.57E−5 ND NA P1B6 0.14 9.72E−5 ND NA P1H80.21 8.99E−5 ND NA P8G4 0.21 2.76E−4 ND NA NA = does not apply; ND = nobinding detected

For exemplary antibodies 1C1 and 3P10, affinity is driven mainly by avery slow off-rate (see, e.g., FIG. 3). Antibodies that bind to GFRAL asshown in Table 26 above do not recognize (e.g., bind to) GNFR alpha 1,the most closely related homolog of GFRAL.

Example 3: Functional Assays

Antibodies to GFRAL generated, for example, such as described in Example1, were tested for their functional activity in cell-based reporterassays.

For example, ELK1-luciferase reporter assays, which measure human GDF15(hGDF15)-induced human GFRAL/RET signaling, were performed usingtransiently transfected HEK293T. The transfecting plasm ids consisted oftwo reporter plasmids, Gal4-Elk1 and 5×UAS-Luc (Agilent TechnologiesPathDetect Elk1 trans-reporting system Cat# 219005), and plasm idsencoding human GFRAL (hGFRAL), cynomolgus monkey GFRAL (cynoGFRAL),mouse GFRAL (mGFRAL), or rat GFRAL (rGFRAL), and human RET (hRET),cynomolgus monkey RET (cynoRET), mouse RET (mRET) or rat RET (rRET). Inthese assays, hGDF15-induced activation of recombinantly expressedGFRAL/RET receptor complex in the cells triggers intracellular signalingtransduction, which leads to ERK and then Elk1 phosphorylation. OnceGal4-Elk1 is phosphorylated, Gal4-Elk1 binds to the 5×UAS promoterregion and turns on luciferase reporter gene transcription. The activityof luciferase is then measured in luciferase enzymatic assays.

Representative results for antibodies to GFRAL inhibiting humanGFRAL/RET signaling are shown in Table 27 below.

TABLE 27 IC₅₀ (nM) Clone ID hGFRAL/hRET mGFRAL/mRET 5F12 0.834 6.2763P10 4.088 1.029 17J16 0.6658 0.4075 6G9 2.65 ND 2B8 1.923 2.449 6N161.618 ND 8C10 1.786 WB 2B11 7.896 ND 25M22 2.887 1.113 12A3 4.993 0.93341B3 4.136 ND 19K19 3.877 0.8948 1C1 8.638 1.189 8D8 2.106 1.419 22N57.744 2.599 2A9 3.706 7.871 2B3 7.124 7.761 24G2 18.94 8.324 5A20 3.190.6 2I23 7.185 NP 1A3 ND NP P1B6 ND NP P1H8 ND NP P8G4 ND NP ND =blocking not detected in assay NP = assay not performed WB = weakblocking detected in assay

For some experiments, the above mentioned four plasmids (e.g., 2reporter plasmids, GFRAL, RET) were transfected into newly harvestedcells in suspension using FuGene6 transfection reagent (Promega). TheGFRAL and RET DNA ratio in transfection was optimized for the each pairof receptors from indicated species and varied between 12:1 to 60:1.Transfected cells were seeded into 384-well plate (7500 cells/25μL/well) in normal growth medium. After overnight incubation at 37° C.,a mix of serially diluted antibodies and fixed concentration of hGDF15were added. After 6 hrs at 37° C. incubation with the antibodies, anequal volume of Bright-Glo reagent (Promega) was added and luminescencesignal was read using Enspire reader (Perkin Elmer).

Simultaneous addition of antibodies, antagonizing the hGDF15 effect,blocked hGDF15 signaling in a dose-dependent manner preventingexpression of luciferase reporter gene.

Example 4: Additional Functional Assays

Anti-hGFRAL antibodies were tested for their hGDF15 antagonisingactivity in an additional cell-based assay, such as an U2OS assay stablyexpressing hGFRAL and hRET. One day before the assay, the cells wereplated in 90 μl of DiscoveRx Assay Complete Cell Plating 16 Reagent(DiscoveRx, Cat#93-0563R16B) at 20K/each of 96 well plate. Next day thecells were treated with a mix of serially diluted antibodies and a fixedconcentration of hGDF15 for 10 minutes at 37° C. Cis-bio Cellul'erkassay kit (Cat# 64ERKPEH) was used to assay for ERK phosphorylationlevel following the manufacturer's protocol. Similar to the Hek293T Elk1reporter assay, hGDF15 antagonising antibodies were able to preventhGDF15-induced phosphorylation in a dose-dependent manner.

Representative results for antibodies to GFRAL preventing hGDF15-inducedphosphorylation are shown in Table 28 below.

TABLE 28 Clone ID IC₅₀ (nM) 5F12 0.06 3P10 0.08 17J16 0.10 6G9 0.17 2B80.22 6N16 0.27 8C10 0.28 2B11 0.46 25M22 0.53 12A3 0.54 1B3 0.57 19K190.61 1C1 0.61 8D8 0.63 22N5 0.90 2A9 0.94 2B3 1.48 24G2 1.86 5A20 1.802I23 NP 1A3 NP P1B6 NP P1H8 NP P8G4 NP NP = assay not performed

Example 5: Ligand Competition, Domain Mapping and Epitope Binning

Antibodies that were selected for binding to GFRAL were evaluated inligand competition binding assays, domain and epitope binningexperiments.

Briefly, ligand competition binding assays were performed by capturingGFRAL-Fc protein or RET and GFRAL Fc-heterodimers onto ELISA plates byanti-human Fc. Plates were blocked using PBS/1% BSA. 15 μL of a dosetitration of antibodies were added to the wells starting at 10 μg/mLwith a 3× dilution and incubated at room temperature for 30 min. Withoutwashing, biotinyated GDF15 (GFRAL ligand) was added to the wells for anadditional 1 hr. After 3 washes, 15 μL of HRP-streptavidin secondarywere added to the wells and incubated at room temperature for 1 hr.After 3 washes, 15 μL of TMB were used to develop the plate. A positivesignal indicates that biotinylated GDF15 still binds to GFRAL capturedon the plate and the antibody is not a ligand competitor. A negativesignal indicates that biotinylated GDF15 no longer binds to GFRALcaptured on the plate and the antibody is a ligand competitor.

Briefly, domain mapping assays were performed by transientlytransfecting 293EXPI cells with nucleic acid sequences encoding GFRAL(SEQ , GFRAL domain 1, GFRAL domain 2, GFRAL domain 1+2, GFRAL domain1+3, GFRAL domain 2+3, or GFRAL domain 3.

Seven GFRAL deletion constructs (Constructs 1 to 7) were tested, whichincluded the following features contiguously from N-terminus toC-terminus: IgK signal sequence (depicted by lower case and underlinedletters in the below sequences), FLAG tag sequence (depicted by lowercase and italicized letters in the below sequences) and GFRAL proteinsequences with various extracellular domain combinations (domain 1depicted in bold capital letters; domain 2 depicted in underlinedcapital letters; domain 3 depicted in bold and underlined letters).Construct 1 (IgK-2Flag-GFRAL; SEQ ID NO: 1817) contained human GDF15polypeptide (residues Q20 to L394), in which domains 1, 2 and 3 werepresent. Construct 2 (IgK-2Flag-GFRAL domain 1; SEQ ID NO: 1818)contained GFRAL-ΔD2,ΔD3 (residues Q20 to S130 and F317 to L394), inwhich domains 2 and 3 were deleted. Construct 3 (IgK-2Flag-GFRAL domain2; SEQ ID NO: 1819) contained GFRAL-ΔD1,ΔD3 (residues S121 to C210 andF317 to L394), in which domains 1 and 3 are deleted. Construct 4(IgK-2Flag-GFRAL domain 1+2; SEQ ID NO: 1820) contained GFRAL-ΔD3(residues Q20 to C210 and F317 to L394), in which domain 3 was deleted.Construct 5 (IgK-2Flag-GFRAL domain 1+3; SEQ ID NO: 1821) containedGFRAL-ΔD2 (residues Q20 to S130 and C220 to L394), in which domain 2 wasdeleted. Construct 6 (IgK-2Flag-GFRAL domain 2+3; SEQ ID NO: 1822)contained GFRAL-ΔD1 (residues S121 to L394), in which domain 1 wasdeleted. Construct 7 (IgK-2Flag-GFRAL domain 3; SEQ ID NO: 1823)contained GFRAL-ΔD1, ΔD2 (residues A211 to L394), in which domains 2 and3 were deleted.

Construct 1 (IgK-2Flag-GFRAL; SEQ ID NO: 1817) mdmrvpaqllgllllwlrgarcdykddddksaggdykddddkgg QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGF KGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPT CLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC FNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRT SRISSKARDPSSIQIPGELConstruct 2 (IgK-2Flag-GFRAL domain 1; SEQ ID NO: 1818)mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkgg QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL Construct 3 (IgK-2Flag-GFRAL domain 2;SEQ ID NO: 1819) mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkggSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKA RDPSSIQIPGELConstruct 4 (IgK-2Flag-GFRAL domain 1 + 2; SEQ ID NO: 1820)mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkgg QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGF KGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSK ARDPSSIQIPGELConstruct 5 (IgK-2Flag-GFRAL domain 1 + 3; SEQ ID NO: 1821)mdmrvpaqllgllllwlrgar cdykddddksaggdykddddkgg QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRK SCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL Construct 6 (IgK-2Flag-GFRAL domain 2+ 3; SEQ ID NO: 1822) mdmrvpaqllgllllwlrgarcdykddddksaggdykddddkggSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPT CLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC FNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTS RISSKARDPSSIQIPGELConstruct 7 (IgK-2Flag-GFRAL domain 3; SEQ ID NO: 1823)mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkggAVNMVP PPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL

Cells were incubated with 1 μg/mL of antibodies for 30 mins at 4° C.After washing, cells were incubated with a fluorochrome labeledanti-mouse Fc secondary antibody either A488 or A647 for 30 mins at 4°C. After washing, cells were analyzed by FACs. A positive signalindicates that the antibody binds to the domain overexpressed by thetransfected 293EXPI cells. A negative signal indicates that the antibodydoes not bind to the domain overexpressed by the transfected 293EXPIcells.

Briefly, epitope binning assays were performed by directly coatingplates with 2 ug/mL of antibodies (mAb1). Plates were blocked usingPBS/1% BSA. 2 μg/mL of antibodies (mAb2) were pre-incubated with 50ng/mL of GFRAL-Fc protein for 30 min before adding to wells at roomtemperature for 1 hr. After 3 washes, 15 μL of HRP-anti-human Fcsecondary were added to the wells and incubated at room temperature for1 hr. After 3 washes, 15 μL of TMB were used to develop the plate. Apositive signal indicates that GFRAL-Fc protein still binds to thecaptured antibody (mAb1) on the plate and antibody (mAb2) is not in thesame epitope bin as the captured antibody (mAb1). A negative signalindicates that GFRAL-Fc protein no longer binds to the captured antibody(mAb1) on the plate and antibody (mAb2) is in the same epitope bin asthe captured antibody (mAb1).

Representative results for inhibition of GDF15 binding to GFRAL, domainmapping of GFRAL and epitope binning are shown in Table 29 below.

TABLE 29 Clone ID Inhibition GFRAL Domain Epitope Bin 5F12 No D3 4 3P10No D3 4 17J16 Partial D2 1 6G9 No D3 4 2B8 Yes — 2 6N16 No D3 4 8C10 YesD2 2 2B11 No D3 4 25M22 Yes D2 2 12A3 Yes D2 2 1B3 No D3 4 19K19 Yes D22 1C1 Yes D2 3 8D8 Yes D2 1 22N5 Yes D2 3 2A9 Yes D2 3 2B3 Yes ND 1 24G2Yes NA 1 5A20 Partial D2 1 2I23 No NA 4 1A3 No D1 5 P1B6 No D1 5 P1H8 NoD1 7 P8G4 No D1 6 NA = does not apply; ND = no binding detected

As shown in Table 29 above, at least three classes of anti-GFRALantibodies were identified, including antibodies that block GDF15binding (e.g., competitive antagonists), antibodies that do not blockGDF15 binding (e.g., non-competitive antagonists), and partial blockersof GDF15 binding. Antibodies were also identified as binding to domain1, domain 2 or domain 3 of GFRAL. The antibodies that bound to domain 2were found to either inhibit GDF15 binding to GFRAL or at leastlypartically inhibit this binding. The antibodies that bound to domains 1or 3 did not inhibit GDF15 binding to GFRAL.

An alignment of exemplary anti-GFRAL antibodies assayed in Examples 2-4and described above is shown in FIGS. 4A-4B.

Alignments of VH and VL domains for antibodies that were found to binddomain 1, domain 2 or domain 3 are shown in FIGS. 5A-5F, respectively.

Example 6: Humanization

Humanized anti-GFRAL antibodies were generated, including fromantibodies selected as described in Examples 1-5. Exemplary humanizedantibodies are generated comprising one or more CDRs from Tables 1-24,including, for example, the CDRs for the antibody designated 1C1 (see,e.g., Table 1), for the antibody designated 25M22 (see, e.g., Table 8),for the antibody designated 17J16 (see, e.g., Table 7), for the antibodydesignated 5F12 (see, e.g., Table 4), and for the antibody designated3P10 (see, e.g., Table 2). The sequences of VH and VL regions forexemplary humanized antibodies 1C1, 25M22, 17J16, 5F12, and 3P10 areshown in FIGS. 6A-6B, 7A-7B, 8A-8B, 9A-9B, and 10A-10B.

A number of anti-GFRAL antibodies were selected for sequencing andsubsequently for humanization. An NCBI immunoglobulin (Ig) sequenceblastp search was conducted to identify human germ line sequences withsignificant similarity to mouse VH and VL sequences. Examples forpotential human framework sequences are given in Table 30. Consideringsequence similarities, biophysical properties and potentialimmunogenicity, germ line sequences for IGH and IGK were chosen as humanframework sequences. The germline sequences that were chosen arehighlighted in bold in Table 30. In order to identify the best matchingframework 4 sequence, a similar approach was taken for choosing JH andJK germline sequences, and these chosen sequences are also highlightedin bold in Table 30.

TABLE 30 Human Human Human Human germline germline germline germlinedonor donor donor donor sequence sequence sequence sequence Clone ID forVH for JH for VL for JK 3P10 IGHV1-18 IGHJ6 IGKV1-39 IGKJ4 IGHV1-3 IGHJ1IGKV3-20 IGKJ2 IGHV1-46 IGKV2-30 IGHV1-2 IGK4-1 IGHV1-69 IGK3-11 5F12IGHV1-46 IGHJ1 IGKV4-1 IGKJ4 IGHV1-3 IGHJ3 IGKV2-30 IGKJ2 IGHV1-69IGKV1-39 IGHV1-2 IGKV3-11 IGKV1-5 IGKV1-27 25M22 IGHV5-51 IGHJ6 IGKV2-29IGKJ4 IGHV1-46 IGHJ4 IGKV2-28 IGKJ2 IGHV1-3 IGKV2-18 IGHV1-2 IGKV2-30IGHV1-69 IGKV3-20 IGHV1-18 IGKV1-39 1C1 IGHV4-39 IGHJ4 IGKV3-20 IGKJ4IGHV4-31 IGHJ6 IGKV1-39 IGKJ2 IGHV3-33 IGKV2-30 IGHV4-59 IGKV2-28 17J16IGHV1-2 IGHJ6 IGKV2-30 IGKJ4 IGHV1-46 IGKV2-29 IGKJ2 IGHV1-3 IGKV2-40IGHV1-69 IGKV3-20 IGKV1-39 1A3 IGHV5-51 IGHJ6 IGKV2-28 IGKJ4 IGHV1-69IGHJ4 IGKV2-30 IGKJ2 IGHV1-46 IGKV4-1 IGHV1-2 P1B6 IGHV5-51 IGHJ4IGKV3-11 IGKJ4 IGHV1-69 IGHJ1 IGKV1-9 IGKJ2 IGHV1-46 IGKV1-5 IGHV1-2IGKV3-15 IGKV1-33 IGKV3-20 P1H8 IGHV5-51 IGHJ6 IGHV2-28 IGKJ4 IGHV1-69IGHJ4 IGHV2-30 IGKJ2 IGHV1-46 IGHV4-1 IGHV1-2 P8G4 IGHV4-59 IGHJ4IGKV3-11 IGKJ1 IGHV3-23 IGHJ6 IGKV1-39 IGKJ2 IGHV4-4 IGKV1-5 IGHV3-30IGKV3-20

The CDR sequences of murine antibodies were then transferred (e.g.,grafted) to the corresponding positions of human IGH and IGK andJ-region residues corresponding to framework four were added. Theresulting protein sequence was back translated into a DNA sequence,codon optimized for expression in mammalian cells and synthesized(GeneArt/LifeTechnologies). Subsequently, the synthesized DNA fragmentwas cloned using In-Fusion technology (Clontech) into pTT5 vector (NRCBiotechnology Research Institute) to create expression ready constructsthat contain a Kozak sequence, the hIgK signal peptide followed by thehumanized variable-region of the desired antibody plus the constantregion of the antibody (hIgG1/hIgK). At the same time individualresidues in the framework regions were selected to be back-mutated tomouse residues in order to retain binding affinities. Specialconsideration was given to Vernier Zone residues. Such variants wereeither created by side directed mutatgenesis (QuickChange, Agilent) orde-novo DNA synthesis. Such expression constructs were then used fortransient protein expression in Expi293 cells (Thermo Fischer), secretedantibodies were purified from tissue culture supernatant and tested forbinding affinities. In order to optimize for binding affinity and lowestnumber of necessary back mutations, a second round of construct designswere typically made followed by antibody expression, purification andmeasurement of binding affinities.

Exemplary humanized anti-GFRAL antibodies include an antibodycomprising: a VH that is SEQ ID NO: 1982 (HC-344e) and VL that is SEQ IDNO: 1997 (LC-344h), a VH that is SEQ ID NO: 1978 (HC-344a) and VL thatis SEQ ID NO: 1992 (LC-344c); a VH that is SEQ ID NO: 1978 (HC-344a) andVL that is SEQ ID NO: 1997 (LC-344h); a VH that is SEQ ID NO: 1985(HC-344h) and VL that is SEQ ID NO: 1997 (LC-344h); a VH that is SEQ IDNO: 1961 (HC-375d) and VL that is SEQ ID NO: 1976 (LC-375j); a VH thatis SEQ ID NO: 1962 (HC-375e) and VL that is SEQ ID NO: 1976 (LC-375j); aVH that is SEQ ID NO: 1964 (HC-375g) and VL that is SEQ ID NO: 1967(LC-375a); or a VH that is SEQ ID NO: 1964 (HC-375g) and VL that is SEQID NO: 1976 (LC-375j). [

Example 7: Selection of Humanized Antibodies

Exemplary humanized anti-GFRAL antibodies identified in Example 6 wereassayed for their binding affinity to human GFRAL. Binding affinitymeasurements were made in Biacore based assays. For example, equilibriumdissociation constant (K_(D)) measurements were carried out withpurified antibodies to evaluate their binding to human GFRAL. Usingmethods described in Example II, anti-mouse Fc antibody (Sigma-Aldrich,St. Louis, Mo.) was immobilized on all four flow cells of a CM5 chipusing amine coupling reagents (GE Healthcare LifeSciences, Piscataway,N.J.). Purified humanized anti-GFRAL antibodies were captured (˜100 RUs)on flow cells 2, 3 and 4 using flow cell 1 as a reference. This wasfollowed by injection of human GFRAL in PBS-P buffer) at a flow rate of70 μL/min and the binding kinetics were evaluated at 25° C.

Representative results for binding affinity (e.g., K_(D) (nM)) ofexemplary humanized anti-GFRAL antibodies (e.g., humanized 3P10 andhumanized 5F12) to human GFRAL are shown in Tables 31 and 32 below.

TABLE 31 Binding Affinity to Humanized anti-GFRAL Human GFRAL AntibodiesK_(D) (pM) 3P10 Hybridoma <10 HC-344a/LC-344c 12 HC-344a/LC-344h <10HC-344h/LC-344c <10 HC-344e/LC-344h <10 HC-344h/LC-344h <10

TABLE 32 Binding Affinity to Humanized anti-GFRAL Human GFRAL AntibodiesK_(D) (pM) 5F12 Hybridoma 38 HC-375d/LC-375a 74 HC-375d/LC-375c 75HC-375d/LC-375g 77 HC-375d/LC-375i 62 HC-375d/LC-375j 46 HC-375e/LC-375a91 HC-375e/LC-375c 92 HC-375e/LC-375g 81 HC-375e/LC-375j 48HC-375h/LC-375g 87 HC-375h/LC-375j 60 HC-375g/LC-375a 28 HC-375g/LC-375j57

Example 8: Functional Assays of Humanized Antibodies

Exemplary humanized anti-GFRAL antibodies described in Examples 6 and 7were tested for their functional activity in cell-based reporter assayssimilar to that described in Example 3.

For example, ELK1-luciferase reporter assays, which measure human GDF15(hGDF15)-induced human GFRAL/RET signaling, were performed usingtransfected U2OS and HEK293T. The transfecting plasmids consisted of tworeporter plasmids, Gal4-Elk1 and 5×UAS-Luc (Agilent TechnologiesPathDetect Elk1 trans-reporting system Cat# 219005), and plasm idsencoding human GFRAL (hGFRAL and human RET (hRET). In these assays,hGDF15-induced activation of recombinantly expressed GFRAL/RET receptorcomplex in the cells triggers intracellular signaling transduction,which leads to ERK and then Elk1 phosphorylation. Once Gal4-Elk1 isphosphorylated, Gal4-Elk1 binds to the 5×UAS promoter region and turnson luciferase reporter gene transcription. The activity of luciferase isthen measured in luciferase enzymatic assays.

Representative results for exemplary humanized antibodies to GFRAL(e.g., 3P10 and 5F12) inhibiting human GFRAL/RET signaling are shown inTable 33 and 34 below and in FIG. 11.

TABLE 33 Humanized anti-GFRAL Antibodies IC₅₀ (nM) 3P10 Hybridoma 4.02HC-344a/LC-344c 7.26 HC-344a/LC-344h 4.63 HC-344h/LC-344c 29.4HC-344e/LC-344h 6.95 HC-344h/LC-344h 5.7

TABLE 34 Humanized anti-GFRAL Antibodies IC₅₀ (nM) 5F12 Hybridoma 5.40HC-375d/LC-375a 3.86 HC-375d/LC-375c 3.28 HC-375d/LC-375g 2.79HC-375d/LC-375i 2.75 HC-375d/LC-375j 3.11 HC-375e/LC-375a 2.81HC-375e/LC-375c 2.20 HC-375e/LC-375g 2.27 HC-375e/LC-375j 4.01HC-375h/LC-375g 3.98 HC-375h/LC-375j 7.29

For some experiments, the above mentioned four plasmids (e.g., 2reporter plasmids, GFRAL, RET) were transfected into newly harvestedcells in suspension using FuGene6 transfection reagent (Promega). TheGFRAL and RET DNA ratio in transfection was optimized for the each pairof receptors from indicated species and varied between 12:1 to 60:1.Transfected cells were seeded into 384-well plate (7500 cells/25μL/well) in normal growth medium. After overnight incubation at 37° C.,a mix of serially diluted antibodies and fixed concentration of hGDF15were added. After 6 hrs at 37° C. incubation with the antibodies, anequal volume of Bright-Glo reagent (Promega) was added and luminescencesignal was read using Enspire reader (Perkin Elmer).

Simultaneous addition of humanized anti-GFRAL antibodies, antagonizingthe hGDF15 effect, blocked hGDF15 signaling in a dose-dependent mannerpreventing expression of luciferase reporter gene.

Example 9: Additional Functional Assays of Humanized Antibodies

Exemplary humanized anti-hGFRAL antibodies were tested for their hGDF15antagonising activity in an additional cell-based assay, such as an U2OSassay stably expressing hGFRAL and hRET, as described in Example 4. Oneday before the assay, the cells were plated in 90 μl of DiscoveRx AssayComplete Cell Plating 16 Reagent (DiscoveRx, Cat#93-0563R16B) at20K/each of 96 well plate. Next day the cells were treated with a mix ofserially diluted antibodies and a fixed concentration of hGDF15 for 10minutes at 37° C. Cis-bio Cellul'erk assay kit (Cat# 64ERKPEH) was usedto assay for ERK phosphorylation level following the manufacturer'sprotocol.

Similar to the Hek293T Elk1 reporter assay described in Example 4,humanized anti-hGFRAL antibodies (e.g., humanized 3P10) were able toprevent hGDF15-induced phosphorylation in a dose-dependent manner.

Representative results for humanized anti-GFRAL antibodies preventinghGDF15-induced phosphorylation are shown in Table 35 below and in FIG.12.

TABLE 35 Humanized anti-GFRAL Antibodies IC₅₀ (nM) 3P10 Hybridoma 5.46HC-344a/LC-344c 5.13 HC-344a/LC-344h 5.02 HC-344h/LC-344c 15.1HC-344e/LC-344h 3.3 HC-344h/LC-344h 2.18

In a control experiment, GFRα1 (bound on chip surface) showed binding toits natural ligand GDNF (in solution, 50nM) (see FIG. 13C).

Exemplary humanized anti-GFRAL antibodies (e.g. HC-344e+LC-344h) did notbind to receptor GFRα1, but showed high affinity binding to hGFRAL(FIGS. 13A and 13B) indicating specificity for GFRAL.

Example 10: Animal Studies

The effects of anti-GFRAL antibodies were evaluated in multiple animalstudies.

A: Anti-GFRAL Antibodies Inhibit GDF15-Induced Weight Loss in DIO Mice(Acute)

To determine if an anti-GFRAL antibody is able to neutralize theGDF15-induced weight lowering effect in diet-induced obesity (DIO) mice,17 week old male C57BL/6J DIO mice were used (Jackson Labs West,Sacramento, Calif.). Mice (41.3 g±0.4 g) were randomly assigned toreceive an exemplary anti-GFRAL antibody (e.g., 1C1, 3P10, 17J16, 5A20,25M22, 5F12, 8D8 and 12A3) or an anti-GDF15 antibody (e.g., 1MO3). Eachgroup had 6 mice per group. Each treatment group had its own PBS controlgroup. The antibody dosage was 20 mg/kg. One day post injections witheither PBS or antibody, the mice then received 3 consecutive dailyinjections of a GDF15 protein (e.g., having an amino acid sequence ofSEQ ID NO:1811) at 0.5 mg/kg. Daily body weight and food intake wererecorded (day 1- 3 and day 7). Sartorius balance LE5201 was used forweighing. An automatic weighing record program (Sartorius YSW05 SoftwareWedge, Sartorius Mechatronics Corporation, 5 Orville Drive, Suite 200Bohemia, N.Y. 11716) was used to transmit the weight data to a MicrosoftExcel spreadsheet automatically.

Data are presented by mean ±sem in term of raw body weight, delta bodyweight change and percentage of body weight change (i.e., % of deltabody weight change over baseline body weight). A Student's t-test wasused (two tails, two ends). In these experiments, P<0.05 was consideredas statistically significant.

Exemplary anti-GFRAL antibodies, 1C1, 3P10, 17J16, 5A20, 25M22, 5F12,8D8 and 12A3, are able to reverse GDF15-induced body weight loss andfood intake reduction (FIGS. 14A and 14B). An anti-GDF15 antibody alsoreversed GDF15-induced body weight loss and food intake reduction inthis model.

To determine if a non-competitive anti-GFRAL antibody or a competitiveanti-GFRAL antibody block exogenous GDF15-induced weight lowering effectin a dose-dependent manner, 19 week old male C57BL/6J DIO mice wereused. Initially mice (43.1 g±0.3 g) were randomly assigned to PBS (8mice per group) or a GDF15 protein (e.g., having an amino acid sequenceof SEQ ID NO: 1811) groups (0.1 mg/kg, 1.0 mg/kg, or 10 mg/kg, 24 miceper group). A GDF15 protein was confirmed to reduce body weight and foodintake dose-dependently in the following day. Furthermore, the GDF15treatment groups (0.1 mg/kg, 1.0 mg/kg, or 10 mg/kg) were randomlyassigned to receive PBS, or an exemplary anti-GFRAL antibody (e.g., 1C1or 3P10) (8 mice per group). Daily body weight and food intake wererecorded (day 1- 3 and day 7).

Exemplary anti-GFRAL antibodies reversed body weight reduction and foodintake reduction that was induced by GDF15 (FIGS. 15 and 16).

B: Anti-GFRAL Antibodies Promote Body Weight Gain in DIO mice (Chronic)

To determine if an exemplary anti-GFRAL antibody is able to increasebody weight independent of exogenous GDF15 protein in a DIO model, 14week old male C57BL/6J DIO mice were used (Jackson Labs West,Sacramento, Calif.). Mice (34.5 g±0.8 g) were randomly assigned toreceive either PBS or exemplary anti-GFRAL antibodies (e.g., 1C1 or3P10) at 10 mg/kg weekly. Each group had 10 mice per group. Daily bodyweight and food intake were recorded for 28 days. Sartorius balanceLE5201 was used for weighing. An automatic weighing record program(Sartorius YSW05 Software Wedge, Sartorius Mechatronics Corporation, 5Orville Drive, Suite 200 Bohemia, N.Y. 11716) was used to transmit theweight data to a Microsoft Excel spreadsheet automatically. Bodycomposition was also evaluated by EchoMRI.

Exemplary anti-GFRAL antibodies increased body weight (by 6% and 7%,respectively) and food intake (by 3.4% and 3.7%, respectively) in DIOMice (FIGS. 17 and 18). The increase in body weight by the exemplaryanti-GFRAL antibodies is mainly attributed to increased fat mass (FIG.19).

C: Anti-GFRAL Antibodies Reverse GDF-15-induced Loss of Body Mass in DIOMice

To determine if an exemplary anti-GFRAL antibody is able to neutralizethe GDF15-induced weight lowering effect in DIO mice, 17 week old maleC57BL/6J DIO mice were used (Jackson Labs West, Sacramento, Calif.) anda recombinant adeno-associated virus (rAAV) expressing GDF15 wasconstructed.

Construction of the rAAV was performed as follows: Polymerase chainreactions (PCR) reagents kits with Phusion® high-fidelity DNA polymerasewere purchased from New England BioLabs (F-530L, Ipswich, MA). The PCRreactions were set up according to manufacturer's instruction. AmplifiedDNA fragments containing an Igk signal peptide followed by a GDF15encoding sequence was digested with restriction enzymes Spe I and Not I(the restriction sites were included in the 5′ or 3′ PCR primers,respectively) and were then ligated with AAV transgene vectors that hadbeen digested with the same restriction enzymes. The vector used forexpression contained a selectable marker and an expression cassettecomposed of a strong eukaryotic promoter 5′ of a site for insertion ofthe cloned coding sequence, followed by a 3′ untranslated region andbovine growth hormone polyadenylation tail.

The DIO mice were subjected to a high fat diet (Research Diets, catalog# D12492NI). The high fat diet contained 60 kcal % fat, 20 kcal %protein and 20 kcal % carbohydrate. The mice were administered aone-time tail vein injection of the rAAV described above or a controlAAV vector expressing green fluorescent protein (GFP). 42 days postinjection with rAAV expressing the GDF15 at 1×10¹⁰, mice wereadministered the exemplary anti-GFRAL antibody (3P10) or a controlantibody (anti-KLH antibody) at a dose of 3 mg/kg. There were 20 miceper group. Mice body weight, lean and fat mass, energy expenditure andfood intake were monitored over 12 weeks.

As shown in FIG. 20, the exemplary anti-GFRAL antibody (3P10) reversedthe GDF15-induced loss of body weight and loss of fat and lean mass inDIO mice. Additionally, as shown in FIG. 21, the exemplary anti-GFRALantibody reversed the GDF15-induced increase in energy expenditure andGDF15-induced reduction in food intake.

D: Anti-GFRAL Antibodies Reverse GDF-15-induced Loss of Body Mass inLean Mice (Chronic)

To determine if an exemplary anti-GFRAL antibody is able to neutralizethe GDF15-induced weight lowering effect in lean mice, 15 week old maleC57BL/6J mice were used (Jackson Labs West, Sacramento, Calif.). Themice were placed on a Chow Diet. As described in Example 11, part c, themice were administered a one-time tail vein injection of the rAAVexpressing GDF15 at 1×10¹⁰ or a control AAV vector expressing greenfluorescent protein (GFP). 28 days post injection with rAAV expressingthe GDF15, mice were administered the exemplary anti-GFRAL antibody(3P10) or a control antibody (anti-KLH antibody) at a dose of 3 mg/kg.There were 20 mice per group. Mice body weight, lean and fat mass,energy expenditure and food intake were monitored over 12 weeks.

As shown in FIG. 22, the exemplary anti-GFRAL antibody reversed theGDF15-induced loss of body weight, which included a reversal of bothloss of fat mass and loss of lean mass in lean mice. Additionally, asshown in FIG. 23, the exemplary anti-GFRAL antibody reversed theGDF15-induced change in respitory exchange ratio (RER) and GDF15-inducedreduction in food intake in lean mice.

E: Anti-GFRAL Antibodies Do Not Increase Body Weight Independent ofGDF15-Induced Weight Loss in Lean Mice

To determine if an exemplary anti-GFRAL antibody is able to increasebody weight independent of exogenous GDF15 protein in lean mice, 15 weekold male C57BL/6J mice were used (Jackson Labs West, Sacramento,Calif.). Mice (26.1 g±0.3 g) were randomly assigned to receive eitherPBS or an exemplary anti-GFRAL antibody (e.g., 1C1 or 3P10) at 10 mg/kgweekly. Each group had 10 mice per group. Daily food intake and bodyweight were recorded in the first week, and then recorded weekly foranother 3 weeks. Sartorius balance LE5201 was used for weighing.

Repeat doses of exemplary anti-GFRAL antibodies 1C1 and 3P10 do notsignificantly alter body weight and food intake in lean mice, althoughantibody 3P10 showed a weak trend to increase body weight (FIGS. 24 and25).

F: Anti-GFRAL Antibodies Reverse Body Weight Loss and Increased EnergyExpenditure in Mice with Chronic Kidney Damage

To determine if an exemplary anti-GFRAL antibody is able to reverse lossof body weight and increased energy expenditure in DIO mice with chronickidney damage, 15 week old male C57BL/6J mice were used (Jackson LabsWest, Sacramento, Calif.). After 5 weeks on HFD +adenine diet (0.3% @induction phase for 10 days, then reducing to 0.1° A @ maintainancephase for another 3 weeks), DIO Mice (26.1 g±0.3 g) were randomlyassigned to receive either control antibody (anti-KLH) or exemplaryanti-GFRAL antibodies (e.g., 3P10) at 1 mg/kg weekly. Each group had 12mice per group. Daily body weight was recorded in the first week, andthen recorded weekly for another 8 weeks. Sartorius balance LE5201 wasused for weighing.

As shown in FIG. 26A, in order to verify the effect of adenine on kidneyfunction, 10-day treatment with dietary adenine (0.25% adenine in HFD)was shown to induce kidney damage in mice. Dietary adenine alsoincreased serum GDF15 levels (see FIG. 26B) and promoted weight loss(see FIG. 26C). Additionally, dietary adenine increased energyexpenditure in mice (see FIG. 27A).

As shown in FIG. 27B, the exemplary anti-GFRAL antibody reversed theincreased energy expenditure in mice with chronic kidney damage.Additionally, as shown in FIG. 28, the exemplary anti-GFRAL antibodyreversed the loss of body weight and loss of fat and lean mass in micewith chronic kidney damage.

G: Humanized Anti-GFRAL Antibodies Inhibit GDF15-Induced Weight Loss ina Dose-Dependent Manner

To determine if an humanized anti-GFRAL antibody is able to neutralizethe GDF15-induced weight lowering effect in diet-induced obesity (DIO)mice, 17 week old male C57BL/6J DIO mice were used (Jackson Labs West,Sacramento, Calif.). Initially mice (42 g±0.4 g) were randomly assignedto PBS (8 mice per group) or a GDF15 protein (56 mice per group). AGDF15 protein was confirmed to reduce body weight and food intakedose-dependently in the following day. Furthermore, 8 mice per groupwere randomly assigned to receive either an vehicle (anti-KLH) or anexemplary mouse anti-GFRAL antibody (e.g., m3P10) ata dosage of 1.0mg/kg, or an exemplary humanized anti-GFRAL antibody (e.g.,h3P10=HC-344e+LC-344h) at dosages of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3mg/kg or 10 mg/kg. Daily body weight was recorded (day 1-7). Sartoriusbalance LE5201 was used for weighing. An automatic weighing recordprogram (Sartorius YSW05 Software Wedge, Sartorius MechatronicsCorporation, 5 Orville Drive, Suite 200 Bohemia, N.Y. 11716) was used totransmit the weight data to a Microsoft Excel spreadsheet automatically.

Data are presented by mean±sem in term of delta body weight change. AStudent's t-test was used (two tails, two ends).

Exemplary humanized anti-GFRAL antibody HC-344e+LC-344h is able toinhibit GDF15-induced body weight loss in a dose-dependent manner (FIG.29).

Example 11: Crystal Structure of GFRAL/GDF15 Complex

A: Complex Formation and Crystallization

A complex of a GFRAL protein and a GDF15 protein was made by mixing 1.2molar excess of a GFRAL (W115-E351) protein with 1 molar GDF15 proteinsubunit (0.5 molar GDF15, which is a homodimer of two GDF15 subunitslinked by a pair of disulfide bonds). The complex was purified by sizeexclusion chromatography to remove excess GFRAL. The GFRAL/GDF15 complexwas crystallized by mixing 1 μL protein at 5 mg/ml with 0.5 μL reservoirsolution and 0.5 μL seed in a crystallization drop, with the reservoirsolution containing 1.0 mL of 0.1 M Bis-Tris pH 6.0, 1.5 M (NH₄)₂SO₄ and10% ethylene glycol. The seed crystals were obtained from acrystallization condition including a reservoir solution of 0.1 MBis-Tris pH 6.0 and 1.5 M (NH₄)₂SO₄. The crystallization setup was keptat room temperature in Rigaku 24 well clover leaf plate. Thecrystallization drop showed small needle crystals after three days ofincubation.

An exemplary small needle crystal of a comples of a GFRAL protein and aGDF15 protein is shown in FIG. 30.

The molecular model was not available for GFRAL, hence NaBr soaking wasused to determine crystal phasing. A GFRAL/GDF15 crystal obtained asdescribed above was soaked with 0.5 M NaBr and 0.75 M NaBr containingreservoir solution. After 30 minutes, 0.5 M NaBr soaked crystals were ingood condition, whereas 0.75 M NaBr soaking yielded cracked crystals.Crystals from both soaks and un-soaked crystals were mounted with 30% EGas a cryo-protectant.

The model described herein provides the first structural information fora GFRAL protein and the binding of a GFRAL protein to a GDF15 protein.

B: Data Collection and Structure Determination

GFRAL/GDF15 complex crystals were obtained and harvested from a 0.1 MBis-Tris pH 6.0, 1.5 M (NH₄)₂SO₄ and 10% ethylene glycol reservoircondition as soaked and unsoaked crystals from 0.5 M and 0.7 M NaBrsoaks. The crystals were treated with the mother liquor supplementedwith 20% ethylene glycol as cryoprotectant and flash-frozen in liquidnitrogen. These crystals were then examined for x-ray diffraction at thesynchrotron beamline IMCA-CAT, Advanced Photon Source, Argonne NationalLab (ALS). The crystal diffracted up to 2.28-2.20 Å resolution.

X-ray diffraction statistics for exemplary GFRAL/GDF15 complex crystalsare shown in Table 36.

TABLE 36 Data collection statistics Crystal I Wave length 0.9786 Å Spacegroup P2₁ Unit cell (Å) a = 75.352 b = 88.768 c = 121.293 Resolution(Å)^(†) 50-2.20 (2.28-2.20) Number of measurements 118,710 Number ofunique reflections  20,379 R_(sym) (%)^(†) 0.09 (0.58) Completeness(%)^(†) 97.4 (85.4) I/σ^(†) 18.9 (2.2) Redundancy^(†) 5.8 (4.9)Molecules in the A.U. 1 GFRAL 1 GDF15 ^(†)The parenthesis is for thehighest resolution shell in Å.

Molecular replacement of GFRAL/GDF15 was performed by using the scaleddataset with a previously solved GFRAL/GDF15 complex at 3.2 Åresolutions as a starting model and the rigid body refinement (SeeVagin, A. A., et al., (2004) “REFMAC5 dictionary: Organization of priorchemical knowledge and guidelines for its use.” Acta Crystallogr. D60:2284-2295) and initial positional refinement was completed in REFMAC5as implemented in CCP4. Several rounds of model rebuilding resulted instructures of the GFRAL/GDF15 complex.

Exemplary structures of a complex of a GFRAL protein and a GDF15 proteinare shown, for example, in FIG. 31-39B.

Inspection of the initial electron density maps showed unambiguousdensity for GFRAL and GDF15. After rigid body refinement, several roundsof model building and restrained refinement were performed using COOT(See Emsley, P. and Cowtan, K. (2004) “COOT: model-building tools formolecular graphics.” Acta Crystallogr. D 60:2126-2132). After placementof the solvent molecules final refinement was completed.

The atomic coordinates from the x-ray diffraction patterns for theGFRAL/GDF15 complex are found in Table 49.

Refinement statistics for exemplary crystals are shown in Table 37.

TABLE 37 Refinement Statistics Refinement Range (Å) 35.82-2.20 R_(cryst)(%) 20.1 R_(free) (%) 26.2 Molecules GDF15, GFRAL; Water molecules 1, 1;132 Bond lengths (Å)   0.019 Bond angles (°)   1.943 Average B-factors(Å²) Overall Main chain atoms (GDF15, GFRAL) 43.9, 57.6 Side chain atoms(GDF15, GFRAL) 51.2, 66.8 Water molecules 56.7 Ramachandran Plot (%)Overall Favored 96.3 Allowed  2.7 Disallowed  1.0

The clear electron density for GFRAL in an exemplary GFRAL/GDF15 complexcrystal is illustrated in FIG. 31. FIG. 31 shows an electron density map(2fo-fc) for the GFRAL molecule calculated with 2.20 A resolution dataand contoured at the 1σ level. The GFRAL residues are clearly visible.

C: Crystal Structure of GFRAL/GDF15 Complex

The crystal structure of a complex of a GFRAL protein and a GDF15protein was determined.

Core interaction interface amino acids were determined as being theamino acid residues (on a protein such as GFRAL) with at least one atomless than or equal to 4.5 Å from the GFRAL interacting proteins (such asGDF15). 4.5 Å was chosen as the core region cutoff distance to allow foratoms within a van der Waals radius plus a possible water-mediatedhydrogen bond.

Boundary interaction interface amino acids were determined as the aminoacid residues (on a protein such as GFRAL) with at least one atom lessthan or equal to 5 Å from core interaction interface amino acids onGFRAL that interact with GFRAL interacting proteins (such as GDF15).Less than or equal to 5 Å was chosen as the boundary region cutoffdistance because proteins binding to residues less than 5 Å away fromcore interaction interface amino acids on GFRAL will be within the vander Waals radius of GFRAL interacting proteins.

Amino acids that met these distance criteria were calculated with theMolecular Operating Environment (MOE) program from CCG (ChemicalComputing Group).

FIG. 32 shows an exemplary illustration of a heterodimeric GFRAL/GDF15complex, as found in the asymmetric unit of a GFRAL/GDF15 proteincrystal. The dimeric molecule GDF15 has one intermolecular disulfidelink, which was found to be weak due to radiation damage. One side of aGDF15 molecule can form a dimer in the asymmetric unit. FIG. 33 shows anexemplary dimeric arrangement of the GFRAL/GDF15 hetero dimers in aGFRAL/GDF15 crystal.

FIG. 34A-34B illustrate the extent of the protein-protein contacts on aGFRAL-GDF15 interface. The contact region on GFRAL is indicated by lightgray arrows; the contact region on GDF15 is indicated by the blackarrows.

FIG. 35 shows that three a-helices of GFRAL are involved in aGFRAL/GDF15 interface. Multiple disulfide bridges appear to stabilizethe structural arrangement of the three GFRAL α-helices.

FIGS. 36A-36D illustrate different aspects of a GFRAL/GDF15 interfaceand the core and boundary amino acid residues of a GFRAL protein and aGDF15 protein involved in forming a GFRAL/GDF15 interface. The GFRALprotein and the GDF15 protein are depicted as ribbon diagrams withresidues in the GFRAL/GDF15 interface shown in a space-filled surfacerepresentation. FIGS. 36A-36C show core interaction interface aminoacids of the GFRAL protein and the GDF15 protein. FIG. 36D showsboundary interaction interface amino acids.

The amino acid sequence of a full-length precursor human GFRAL proteinis shown below:

GFRAL sequences SEQ ID NO: 1797        10         20         30         40 MIVFIFLAMG LSLENEYTSQTNNCTYLREQ CLRDANGCKH         50         60         70         80AWRVMEDACN DSDPGDPCKM RNSSYCNLSI QYLVESNFQF        90        100        110        120 KECLCTDDFY CTVNKLLGKKCINKSDNVKE DKFKWNLTTR        130        140        150        160SHHGFKGMWS CLEVAEACVG DVVCNAQLAS YLKACSANGN       170        180        190        200 PCDLKQCQAA IRFFYQNIPFNIAQMLAFCD CAQSDIPCQQ        210        220        230        240SKEALHSKTC AVNMVPPPTC LSVIRSCQND ELCPRHYRTF       250        260        270        280 QSKCWQRVTR KCHEDENCISTLSKQDLTCS GSDDCKAAYI        290        300        310        320DILGTVLQVQ CTCRTITQSE ESLCKIFQHM LHRKSCFNYP       330        340        350        360 TLSNVKGMAL YTRKHANKITLTGFHSPFNG EVIYAAMCMT        370        380        390 VTCGILLLVMVKLRTSRISS KARDPSSIQI PGEL

GFRAL amino acids at the interface of the GFRAL/GDF15 complex are shownin Table 38.

TABLE 38 GFRAL Residues Binding GDF15* Core interaction Boundaryinteraction interface amino acids interface amino acids GLY140 SER156LEU148 GLN147 ALA149 LEU148 ALA146 ALA149 VAL142 SER150 ASN145 TYR151VAL139 LEU152 ALA135 LYS153 GLU136 ALA154 LEU152 CYS155 LEU132 PHE174SER201 TYR175 ALA204 GLU136 LEU205 ALA137 LYS153 CYS138 ILE196 VAL139PRO197 GLY140 GLN200 ASP141 VAL142 VAL143 CYS144 ASN145 ALA146 LEU186CYS189 CYS191 ALA192 GLN193 SER194 ASP195 ILE196 PRO197 CYS198 GLN199GLN200 SER201 LYS202 GLU203 ALA204 LEU205 HIS206 SER207 SER130 CYS131LEU132 GLU133 VAL134 ALA135 *GFRAL amino acid numbering according to SEQID NO: 1797

The amino acid sequence of mature human GDF15 is shown below:

(SEQ ID NO: 1811) ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPSQFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI

GDF15 residues at the interface of the GFRAL/GDF15 complex are shown inTable 39.

TABLE 39 Residues on GDF15 that bind to GFRAL* Core interaction Boundaryinteraction interface amino acids interface amino acids SER35 SER35LEU34 VAL33 THR94 LEU34 GLY95 ASP93 GLN40 THR94 VAL96 GLY95 LEU98 VAL39PRO36 GLN40 VAL87 ARG37 LEU88 GLU38 ILE89 VAL96 ASP102 SER97 THR100LEU98 PRO85 PRO36 MET86 VAL87 LEU88 ILE89 VAL41 GLN90 LYS91 THR92 THR42LEU104 LEU105 TYR101 ASP102 ASP103 GLN99 THR100 LEU24 TRP32 TRP29 ARG21THR19 TYR83 ASN84 PRO85 MET86 VAL20 *GDF15 amino acid numberingaccording to SEQ ID NO: 1811

D: Model of GFRAL/RET/GDF15 Complex

The RET/GFRα1/GDNF ternary complex described by Goodman et al. (2014)CELL REPORTS 8, 1894-1904 (PDB 4UX8) was used as a template to build amodel of the complex of GFRAL/GDF15 /RET (from GFRAL/GDF15 structure,see, e.g., Examples 11-13). The RET/GFRα1/GDNF template resulted from anelectron microscopy reconstruction of a reconstituted mammalianRET(ECD)-GDNF-GFRα1 ternary complex (Goodman et al., supra).

To compare the structural similarity of the GFRAL/GDF15 crystalstructure from Example 12 and the structure of GFRα1/GDNF in theRET/GFRα1/GDNF template, the GFRAL structure in GFRAL/GDF15 crystal wassuperposed with GFRα1 in GFRα1/GDNF/RET model (PDB 4UX8) using MOE fromCCG. The high quality of the superposition, and therefore the structuralsimilarity of the GFRAL/GFRα1 and GFRAL/GDF15 complexes was demonstratedby an RMSD of GFRAL/GFRα1 backbone residues of 2.21 Å. This ternarycomplex model, including the GFRAL/GDF15 structure and the RETstructure, was used to map the interactions between GFRAL and RET .

FIGS. 37A-37B illustrate exemplary aspects of the superposition of GFRALand GFRα1 in 4XU8. RMSD of backbone residues was 2.21 Å.

FIGS. 38A-38D illustrate exemplary aspects of the interaction of a GFRALprotein with a RET protein in a RET/GFRAL/GDF15 model. In FIG. 38A,interacting GFRAL and GDF15 residues at the GFRAL/GDF15 interface asmodeled are represented by stick models. In FIG. 38B, theRET-interacting residues on GFRAL are depicted in a space filled surfacemodel. In FIG. 38C, the space filled surface model of the coreinteraction residues are highlighted on GFRAL and RET. In FIG. 38D, thespace filled surface model of the boundary interaction residues arehighlighted on GFRAL and RET.

FIGS. 39A-39B illustrate the core and boundary amino acid residues on aGFRAL protein identified in space filled surface models at the modeledRET interface. In FIG. 39A, core residues on GFRAL as modeled are shownin a darker grey in a space-filled surface model. In FIG. 39B, boundaryresidues on GFRAL as modeled are shown in a lighter grey in a spacefilled surface model.

Based on this modeling, a number of GFRAL residues were identified forinteraction with RET residues, as shown in Table 40A. Additionally, anumber of RET residues were identified for interaction with GFRALresidues, as shown in Table 40B.

TABLE 40A Residues on GFRAL that bind to RET in RET/GFRAL/GDF15 ModelCore interaction Boundary interaction interface amino acids interfaceamino acids GLN246 ILE224 ARG247 ARG225 ARG250 GLN241 LYS251 SER242CYS252 LYS243 ASP255 CYS244 GLU256 TRP245 ASN257 GLN246 CYS258 ARG247ILE259 VAL248 SER260 THR249 THR261 ARG250 LEU262 LYS251 THR297 CYS252GLN298 HIS253 SER299 GLU254 ASP255 GLU256 ASN257 CYS258 ILE259 SER260THR261 LEU262 SER263 LYS264 GLN265 ASP266 LEU267 THR268 THR295 ILE296THR297 GLN298 SER299 GLU300 GLU301 SER302 LEU303 ILE306 PHE307 MET310

TABLE 40B Residues on RET that bind to GFRAL in RET/GFRAL/GDF15 ModelCore interaction Boundary interaction interface amino acids interfaceamino acids GLY74 ASP34 THR75 ALA35 TYR76 TYR36 ARG77 HIS71 THR78 TYR73ASN113 LEU72 ARG114 GLY74 PHE116 TYR76 TYR122 THR75 GLN138 ARG77 ARG144THR78 PRO305 ARG79 ALA306 LEU80 LEU310 LEU109 SER110 VAL111 ARG112ASN113 GLY115 ARG114 PHE116 PRO117 LEU118 THR120 VAL121 TYR122 LEU123LYS124 CYS137 GLN138 TRP139 PRO140 GLY141 CYS142 ALA143 ARG144 VAL145TYR146 PHE147 ARG231 ASP264 ASP300 VAL303 VAL304 PRO305 ALA306 SER307GLY308 GLU309 LEU310 ARG312 VAL311 ASN336

Example 12: Crystal Structures of GFRAL/Antibody Complexes

A1: Complex Formation and Crystallization of GFRAL/3P10/25M22 FabComplex

A complex of a GFRAL protein, a 3P10 Fab and a 25M22 Fab(GFRAL/3P10/25M22 Fab complex) was formed by mixing GFRAL(W115-E351)with Fabs of 3P10 and 25M22 in a 1:1.2:1.2 molar ratio of GFRAL: 3P10Fab: 25M22 Fab. The complex was purified from excess Fab molecules on asize exclusion chromatography column. The GFRAL/3P10/25M22 Fab complexwas concentrated and crystallized as follows.

A volume of 1.5 mL of 3P10 Fab::GFRAL::25M22 Fab complex sample wasconcentrated by centrifugation to about 6.6 mg/mL using 10,000 MWCOCentricon® concentrator. The concentrated sample was immediatelysubjected to crystallization screening using 1 μL protein plus 1 μLreservoir per experiment. A first set of 96 conditions were set up thatcovered a factorial-based formulation sampling for the reservoircontent. The crystallization setups were incubated at room temperature.Hampton Index and PegRx crystallization screens were used in the initialrounds of crystallization. The Hampton Index screen did not yield anypotential hits, whereas the PegRx sreen yielded crystals under thefollowing three crystallization conditions B11, D11, and H8:

B11: 0.1 M MES pH 6.0, 20% PEGMME 2000

D11: 0.1 M Imidazole pH 7,0, 12%, PEG 20,000

H8: 0.1 M TRIS pH 8.0, 16% PEG 10,000, 0.2 M Amonium Acetate.

Exemplary crystals of GFRAL/3P10/25M22 Fab complexes obtained undercrystallization conditions B11, D11, and H8 are shown in FIG. 40.

To further optimize crystals for X-ray diffraction, GFRALI3P10125M22complex was concentrated to 5.0 mg/mL and set up for crystallization andadditional optimization, which led to improved crystals. Some improvedcrystals had a trapezium form. Some of the improved crystals wereharvested, treated with a compatible cryoprotectant, and flash-frozen inliquid nitrogen. Identification and use of a compatible cryoprotectantwas important to maintain sample crystallinity and to collect a highquality X-ray diffraction data set. Improved crystals obtained from acrystallization condition including 0.1 M Imidazole pH 7.0, 12% PEG20,000 (D11) were treated with 35% ethylene glycol. Multiple crystalswere flash-frozen in liquid nitrogen and X-ray diffraction data wascollected at a synchrotron (ALS). One crystal diffracted to about 2.9 Aresolution. A full X-ray diffraction dataset was collected with thiscrystal.

The GFRAL/3P10/25M22 Fab complex crystal structure was determined. Theposition of the GFRAL component was clearly identified by molecularreplacement based on the higher a-helix content in GRAL compared to 3P10and 25M22 Fabs. In the exemplary crystal described here, one GFRAL andtwo Fab components (one 3P10 Fab and one 25M22 Fab) formed a ternarycomplex in the asymetric crystal unit.

Molecular replacement was used to identify Fabs in the GFRAL/3P10/25M22Fab complex. PDB 1F8T, was the closest GFRAL homolog available formolecular replacement analysis (-54% identity and -79% similarity toGFRAL). The structure solution clearly showed all Fab and GFRAL aminoacid positions. The 1F8T GFRAL homolog struture was used as a steppingstone to solve the Fab and GFRAL components of the GFRAL/3P10/25M22terniary complex crystal structure. In this structure, the stoichiometryin the was determined to be 1:1:1 3P10 Fab::GFRAL::25M22 Fab.

A2: Complex Formation and Crystallization of GFRAL/8D8/5f12 Fab Complex

A complex of a GFRAL protein, an 8D8 Fab and a 5F12 Fab (GFRAL/8D8/5F12Fab complex) was formed by mixing GFRAL(W115-E351) with Fabs of 8D8 and5F12 in a 1:1.2:1.2 molar ratio of GFRAL: 8D8 Fab: 5F12 Fab. The complexwas purified from excess Fab molecules on a size exclusionchromatography column. The GFRAL/8D8/5F12 Fab complex was concentratedand crystallized as follows.

GFRAL/5F12/8D8 Fab complex was concentrated by centrifugation using10,000 MWCO Centricon® concentrator. The concentrated sample wasimmediately subjected to crystallization screening using 1 μL proteinplus 1 μL reservoir per experiment. A set of 96 conditions wereinitially set up that cover a factorial-based formulation sampling forthe reservoir content. The crystallization setups were incubated at roomtemperature. Positive leads from initial screens were further optimizedto yield high quality crystals that were amenable to diffractionanalysis. A volume of 1.5 mL of 5F12 Fab::GFRAL::8D8 Fab complex sampleis concentrated by centrifugation to about 7.8 mg/mL using 10000 MWCOCentricon concentrator. Immediately upon completion, this sample issubjected to crystallization screening using 1 μL protein plus 1 μLreservoir per experiment. A set of 96 conditions are initially setupthat cover a factorial-based formulation sampling for the reservoircontent. These setups are incubated at room temperature. Initially,Hampton Index and PegRx crystallization screens were used forcrystallization, Index and PegRx gave crystals in the following threecrystallization conditions C6, E11, and C2:

C6: 0.1 M Bis-Tris pH 6.5, 14% PEG 3350

E11: 1.7 M AmSO4, 0.1 M Bis-Tris pH 6.5, 3% PEG MME 550

C2: 0.1 M Imidazole pH 7.0, 20% Jeffamine 2001.

Preliminary crystals were obtained as shown in FIG. 41.

Optimization of PegRx C6 and C2 crystallizing conditions was conductedby micro seeding technique, which lead to suitable diffractionmono-crystals. These suitable diffraction mono-crystals have a hexagonalbipyramid form obtained in 10% Ethylene glycol with 0.1 M Imidazole pH7.0, 20% Jeffamine 2001 pH 7.0. Some of these crystals were harvested;treated with a compatible cryoprotectant; and flash-frozen in liquidnitrogen. Use of a compatible cryoprotectant is important to maintainthe sample crystallinity, which results in a workable data set. Thecrystals were treated 20% MPD obtained from 0.1 M Imidazole pH 7.0, 20%Jeffamine 2001 pH 7.0.

B1: Data Collection and Structure Determination of GFRAL/3P10/25M22 Fab

Complex

X-Ray diffraction data was collected for twenty crystals of theGFRAL/3P10/25M22 Fab complex at a synchrotron (ALS). The first tencrystals yielded one complete data set with a resolution of 3.17 A. Tenadditional crystals that were further optimized diffracted up 2.9 A, andthis data set was used for structure determination and refinement. TheX-ray diffraction data was indexed using DENZO and subsequentlyintegrated and scaled with SCALEPACK from the program suite HKL2000. SeeOtwinowski Z. and Minor W. “Processing of X-ray Diffraction DataCollected in Oscillation Mode ,” METHODS IN ENZYMOLOGY, Volume 276:Macromolecular Crystallography, part A, p.307-326, 1997,C.W. Carter, Jr.& R. M. Sweet, Eds., Academic Press (N.Y.). The X-ray diffraction of theselected crystal was identified as having an orthorhombic Bravaislattice symmetry. The space group was determined to be P2₁2₁2₁ based onthe systematic absences along (h, 0, 0), (0, k, 0), and (0, 0, I) axes.Analysis of the Matthew's coefficient suggests the crystal's asymmetricunit may accommodate one assembly with approximately 124.4 kDa and acorresponding solvent content of 56%.

X-ray diffraction statistics for exemplary GFRAL/3P10/25M22 Fab complexcrystals are shown in Table 41.

TABLE 41 Data collection statistics Crystal I Wavelength 0.9774 Å Spacegroup P2₁2₁2₁ Unit cell (Å) a = 52.500 b = 116.045 c = 227.048Resolution (Å)^(†) 50-2.91 (3.01-2.91) Number of measurements 227,808Number of unique reflections  31,548 R_(sym) (%)^(†) 0.11 (0.86)Completeness (%)^(†) 100 (100) I/σ^(†) 17.8 (2.7) Redundancy^(†) 7.2(7.2) Molecules in the A.U. 2 Fabs 1 GFRAL ^(†)The parenthesis is forthe highest resolution shell in Å.

Molecular replacement of the GFRAL/3P10/25M22 Fab Complex was performedby using the scaled dataset with the previously solved GFRAL (seeExample 12, part C) and the 79% homologous GFRAL structure of 1F8T asstarting models within the program PHASER. Data extending from 40-3.5 Åresolution gave one GFRAL and one Fab. Molrep yielded one more Fab inthe structure solution. The solution had a complex of one GFRAL and twoFabs in the asymmetric unit. The solution was refined using REFMAC. COOTwas used for model building. The refinement density clearly indicatesmissing loops in Fabs and GFRAL. After placement of a few solventmolecules final refinement was completed.

The atomic coordinates from the x-ray diffraction patterns for theGFRAL/3P10/25M22 Fab Complex are found in Table 50.

Exemplary refinement statistics of a GFRAL/3P10/25M22 Fab Complexcrystal structure are shown in Table 42.

TABLE 42 Refinement Statistics Refinement Range (Å) 47.7-2.9 R_(cryst)(%) 23.6 R_(tree) (%) 31.0 Molecules 3P10 Fab::GFRAL::25M22 Fab 1, 1, 1Bond lengths (Å)   0.011 Bond angles (°)   1.651 Average B-factors (Å²)Overall Main chain atoms ( GFRAL) 66.3 Side chain atoms (GFRAL) 71.6Main chain atoms (3P10) Hc:Lc 92.7; 75.5 Side chain atoms (3P10) Hc:Lc89.8; 78.5 Main chain atoms (25M22 ) Hc:Lc 85.5; 101.9 Side chain atoms(25M22 ) Hc:Lc 83.4; 101.2 Water molecules 67.1 Ramachandran Plot (%)Overall Favored 78.3 Allowed 20.6 Disallowed  1.2

The clear electron densities of CDR regions in each chain of the 3P10and 25M22 Fab fragments in the GFRAL/3P10/25M22 Fab complex areillustrated in FIG. 42 (electron density 2mFo-DFc weighted, 1.0σ contourlevel). The 3P10 and 25M22 chains were identified clearly in theelectron density, with the exception of region 131-137 in the 3P10 heavychain (Chain-H), where a few terminal residues were not identified inthe electron density.

B2: Data Collection and Structure Determination of GFRAL/8D8/5F12 FabComplex

The crystals were examined at the synchrotron at APS. Twenty five suchcrystals were examined for x-ray diffraction. Sixteen crystals were sendfirst time to synchrotron which yielded a diffraction >5 Å and thesecond round of optimized crystals were diffracted up 3.5 Å, which isused for structure determination and refinement. The X-ray diffractiondata were indexed using DENZO and integrated and scaled with SCALEPACKfrom the program suite HKL2000 (Otwinowski and Minor, 1997). The X-raydiffraction of the selected crystal was identified as having anorthorhombic Bravais lattice symmetry. The space group was determined tobe P6₅22 based on the systematic absences along (h, 0, 0) axes. Analysisof the Matthew's coefficient suggests the crystal's asymmetric unit mayaccommodate one assembly with approximately 246.6 kDa and acorresponding solvent content of 61%. The structure solution was checkedwith enantiomeric spacegroup P6₁22, which gave very poor Rfactor andRfree during the rigid body and restraint refinement.

X-ray diffraction statistics for the GFRAL/8D8/5F12 complex crystals areshown in Table 43.

TABLE 43 Data collection statistics Crystal I Wavelength 0.9787 Å Spacegroup P6₅22 Unit cell (Å, °) a = 133.021 b = 133.021 c = 564.819 γ = 120Resolution (Å)^(†) 50-3.55 (3.68-3.55) Number of measurements 524,288Number of unique reflections  37,081 R_(sym) (%)^(†) 0.13 (1.00)Completeness (%)^(†) 100 (100) I/σ^(†) 22.0 (3.4) Redundancy^(†) 14.1(14.6) Molecules in the A.U. 4 Fabs 2 Receptor ^(†)The parenthesis isfor the highest resolution shell in Å.

Molecular replacement of Fabs/Receptor was performed by using the scaleddataset with the previously solved of fab pdb:3IU4 used for 8D8 (89%homologous) and pdb:4M7K used for 5F12(84% homologous) were used as astarting models within the program PHASER using the data extending from40-5.0 Å resolution gave two assembly of one receptor and two Fabs. Thesolution had two receptors and four Fabs complex in the asymmetric unit.The solution was refined used REFMAC and Coot was used for modelbuilding.

The structure solution was checked with enantiomeric spacegroup P6₁22.The Molrep gave two receptors and four Fabs complex. The Molrep modelfurther submitted to Refmac for refinement end up with very poorR_(factor) and R_(free) during the rigid body and restraint refinementwhich confirms that the current space group is P6₅22.

The atomic coordinates from the x-ray diffraction patterns for theGFRAL/8D8/5F12 Fab Complex is found in Table 51.

Exemplary refinement statistics of a GFRAL/8D8/5F12 Fab Complex crystalstructure are shown in Table 44.

TABLE 44 Refinement Statistics Refinement Range (Å) 49.2-3.55 R_(cryst)(%) 25.1 R_(free) (%) 35.8 Molecules 3P10 Fab::GFRAL::25M22 2, 2, 2 Bondlengths (Å) 0.011 Bond angles (°) 1.63

The clear electron density of one GFRAL protein with the 8D8 and 5F12Fab fragments (two Fab chains) in the GFRAL/8D8/5F12 Fab complex isillustrated in FIG. 43 (electron density 2mFo-DFc weighted, 1.0σ contourlevel). The refinement density clearly indicates most of the loops inFabs and receptor molecule. The 5F12 and 8D8 chains were identifiedclearly in the electron density except for the region between aminoacids 139-142 in chain-I (5F12 vH), 132-136 in chain-R (8D8 vH) and131-136 in chain-U (8D8 vL), as well as a few terminal residues were notidentified in the density.

C1: Crystal Structure of GFRAL/3P10/25M22 Fab Complex

The crystal structure of a 3P10 Fab::GFRAL::25M22 Fab complex wasdetermined.

3P10 Fab and 25M22 Fab animo acid sequences are shown below, with VH andVL sequences bolded and CDR regions bolded and underlined.

3P10 Fab Hc: (SEQ ID NO: 1824) QIQLVQSGPELKKPGETVKISCKASGYTFTDYGVIWVKQAPGKALKWMGW INTYTGEPTYADDLKG RFAFSLETSASSASLQINNLKNEDTATYFCARRYGPEDIDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVD 3P10 Fab Lc: (SEQ ID NO: 1825)DIVLTQSPVSLAVSLGQRATISCRASESVDNYGISFMS WFQQKPGQPPKL LIYAASHQGSGVPARFSGSGSGTDFSLNIHPMEEDDSAMYFCLQSKEVPW TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC25M22 Fab Hc: (SEQ ID NO: 1826) QVQLQQSGPDLVKPGASVKISCKASGYTFTSYWVNWMKQRPGKGLEWIGR IYPGDGDTNYNGKFKG KATLTADKSSSTAYMQLSSLTSEDSAVYFCARAYLLRLRRTGYYAMDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVD 25M22 Fab Lc: (SEQ ID NO: 1827)DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDEFTYLD WYLQKPGQSPQ LLIFLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

FIG. 44 illustrates an aspect of a 3P10 Fab::GFRAL::25M22 Fab complex bya ribbon diagram. The Fab fragments interact with an asymmetric unit ofGFRAL in the 3P10 Fab::GFRAL::25M22 Fab complex crystal. The crystalstructure also showed that GFRAL epitope residues 290-312 were presentedto the 3P10 antibody and GFRAL N-terminal epitope residues 130-157 werepresented to the 25M22 antibody heavy chain CDR regions.

FIG. 45 illustrates CDR regions of 3P10 and 25M22 Fabs with interactingGFRAL and Fab residues highlighted in boxes.

FIGS. 46-48 illustrate aspects of GFRAL interactions with 3P10 and25M22L Fabs in ribbon diagrams with select amino acid residues shown asstick models. For example, FIG. 46 illustrates aspects of a GFRALepitope in the vicinity of the 3P10 heavy chain CDR region. The CDRsequences of 3P10 Hc are shown in FIG. 45. As another example, FIG. 47illustrates aspects of a GFRAL epitope in the vicinity of the 3P10 lightchain CDR region. The CDR sequences of 3P10 Lc are shown in FIG. 45. Asanother example, FIG. 48 illustrates aspects of a GFRAL epitope in thevicinity of the 25M22 heavy chain CDR region. The CDR sequences of 25M22Lc are shown in FIG. 45.

An analysis of the GFRAL epitope presented to 25M22 Fab in the 3P10Fab::GFRAL::25M22 Fab complex crystal structure showed that 25M22'smechanism of action is that of a competitive GDF15 inhibitor andinvolves blocking GDF15 binding to GFRAL. Exemplary core interactioninterface amino acids on the GFRAL protein and on the 25M22 Fab CDRs forthe heavy and light chains are shown in FIG. 49.

FIG. 50A and 50B illustrates the core amino acids residues in theGFRAL/25M22 Fab interaction interface. FIG. 50A illustrates thestructure of GFRAL with the core 25M22 interaction interface amino acids(binding epitope) on GFRAL highlighted in a space-filled surface model.Core interface amino acids on 25M22 CDRs for GFRAL are also highlightedin a space-filled surface model. FIG. 50B illustrates the structure ofGFRAL in a ribbon diagram with core 25M22 Fab interaction interfaceamino acids on GFRAL (GFRAL epitope residues) highlighted in aspace-filled surface model.

FIG. 51 illustrates boundary interaction interface amino acids on GFRALfor GFRAL/25M22 Fab binding. Boundary of interaction interface aminoacids on GFRAL (for 25M22 Fab binding) are highlighted as space-filledsurface models.

FIGS. 52-53 illustrate the overlap of GFRAL epitopes for 25M22 Fab andGDF15 binding on GFRAL. Two side views of a GFRAL ribbon diagram areshown in the left and right panels of FIG. 52. FIG. 53 shows a top viewof the overlapping GFRAL epitopes for 25M22 Fab and GDF15 binding. Coreinteraction interface amino acids residues on GFRAL for 25M22 Fab andGDF15 binding are highlighted in a space-filled surface model. Lightgray suface shading represents GFRAL surface covered by 25M22 Fab,whereas drak gray surface shading (highlighted by black arrows) showsGFRAL surface covered by GDF15.

GFRAL amino acids at the interface of the GFRAL/25M22 Fab complex areshown in Table 45. To compare the interaction interface amino acids inGFRAL/25M22 Fab and GFRAL/GDF15 complexes, Table 45 further lists theGFRAL amino acids at the interface of the GFRAL/GDF15 complex, which isalso shown in Table 38. Table 45 illustrates that all core interactioninterface amino acid residues on GFRAL that bind to GDF15 are also coreinteraction interface amino acids in the GFRAL/25M22 Fab interaction.

The amino acid sequence of a full-length precursor human GFRAL proteinis shown below (see also Example 12, part C):

GFRAL sequences SEQ ID NO: 1797        10         20         30         40 MIVFIFLAMG LSLENEYTSQTNNCTYLREQ CLRDANGCKH         50         60         70         80AWRVMEDACN DSDPGDPCKM RNSSYCNLSI QYLVESNFQF        90        100        110        120 KECLCTDDFY CTVNKLLGKKCINKSDNVKE DKFKWNLTTR        130        140        150        160SHHGFKGMWS CLEVAEACVG DVVCNAQLAS YLKACSANGN       170        180        190        200 PCDLKQCQAA IRFFYQNIPFNIAQMLAFCD CAQSDIPCQQ        210        220        230        240SKEALHSKTC AVNMVPPPTC LSVIRSCQND ELCRRHYRTF       250        260        270        280 QSKCWQRVTR KCHEDENCISTLSKQDLTCS GSDDCKAAYI        290        300        310        320DILGTVLQVQ CTCRTITQSE ESLCKIFQHM LHRKSCFNYP       330        340        350        360 TLSNVKGMAL YTRKHANKITLTGFHSPFNG EVIYAAMCMT        370        380        390 VTCGILLLVMVKLRTSRISS KARDPSSIQI PGEL

TABLE 45 Residues on GFRAL that bind to GDF15 Residues on GFRAL thatbind to 25M22 Core interaction Core interaction Boundary interactioninterface amino acids interface amino acids interface amino acids LEU132LEU132 SER130 ALA135 ALA135 CYS131 GLU136 GLU136 LEU132 VAL139 VAL139GLU133 GLY140 GLY140 VAL134 VAL142 VAL142 ALA135 ASN145 ASN145 GLU136ALA146 ALA146 ALA137 LEU148 LEU148 CYS138 ALA149 ALA149 VAL139 LEU152LEU152 GLY140 LYS153 LYS153 ASP141 ILE196 ILE196 VAL142 PRO197 PRO197VAL143 GLN200 GLN200 CYS144 SER201 SER201 ASN145 ALA204 ALA204 ALA146LEU205 LEU205 GLN147 LEU148 ALA149 SER150 TYR151 LEU152 LYS153 ALA154CYS155 SER156 PHE174 TYR175 LEU186 CYS189 CYS191 ALA192 GLN193 SER194ASP195 ILE196 PRO197 CYS198 GLN199 GLN200 SER201 LYS202 GLU203 ALA204LEU205 HIS206 SER207 *GFRAL amino acid numbering according to SEQ ID NO:1797

An analysis of the GFRAL epitope presented to 3P10 Fab in the 3P10Fab::GFRAL::25M22 Fab complex crystal structure showed that 3P10'smechanism of action is that of a non-competitive GDF15 inhibitor anddoes not involve blocking GDF15-binding to GFRAL. Exemplary coreinteraction interface amino acids on the GFRAL protein and on the 3P10Fab CDRs for the heavy and light chains are shown in FIG. 54.

FIGS. 55A-55B illustrate the interaction interface residues on GFRAL and3P10 Fab as stick models (FIG. 55A) or a space-filled surface models(FIG. 55B).

FIGS. 56A-56B illustrates core (FIG. 56A) and boundary (FIG. 56B)interaction interface amino acids on GFRAL for 3P10 Fab binding (GFRALstructural 3P10 binding epitope) in a space-filled surface model.

FIG. 57 illustrates the partial overlap of GFRAL epitopes for 3P10 Faband RET on GFRAL. Two side views of a GFRAL ribbon diagram are shown inthe left and right panels of FIG. 57. Interaction interface amino acidsresidues on GFRAL for 3P10 Fab and RET binding are highlighted in aspace-filled surface model.

FIG. 58 illustrates the overlap of GFRAL epitopes for 25M22 Fab and25M22 Fab binding on GFRAL. Top and a bottom views of a GFRAL ribbondiagram are shown on the left (top view) and right (bottom view) panelsof FIG. 58. Boundary interaction interface residues on GFRAL involved in25M22 Fab and 25M22 Fab binding are shown as space-filled surfacemodels.

GFRAL amino acids at the interface of the GFRAL/3P10 Fab complex areshown in Table 46. To compare the interaction interface amino acids inGFRAL/3P10 Fab and GFRAL/RET complexes, Table 446 further lists theGFRAL amino acids at the interface of the GFRAL/RET complex, which isalso shown in Table 40A. Table 46 shows core interaction interface aminoacid residues on GFRAL that bind to both RET and 3P10 Fab in bold.

TABLE 46 Residues on GFRAL that bind to RET in RET/GFRAL/GDF15 Residueson GFRAL that bind to Model 3P10 in 3P10/GFRAL structure Coreinteraction Core interaction Boundary interaction interface amino acidsinterface amino acids interface amino acids Gln246 MET214 LEU164 Arg247PRO216 LYS208 Arg250 PRO217 VAL212 Lys251 GLN290 ASN213 Cys252 CYS291MET214 Asp255 THR292 VAL215 Glu256 CYS293 PRO216 Asn257 ARG294 PRO217Cys258 THR295 PRO218 Ile259 ILE296 THR219 Ser260 THR297 CYS220 Thr261GLN298 LEU221 Leu262 SER299 VAL223 Thr297 GLU301 TRP245 Gln298 LYS305LEU267 Ser299 GLN308 CYS269 HIS309 GLN288 HIS312 VAL289 SER315 GLN290CYS291 THR292 CYS293 ARG294 THR295 ILE296 THR297 GLN298 SER299 GLU300GLU301 SER302 LEU303 CYS304 LYS305 ILE306 PHE307 GLN308 HIS309 MET310LEU311 HIS312 ARG313 LYS314 SER315 CYS316 PHE317 Core interactioninterface amino acid residues on GFRAL that bind to both RET and 3P10Fab are shown in bold.

C1: Crystal Structure of GFRAL/8D8/5F12 Fab Complex

The crystal structure of a 8D8 Fab::GFRAL::5F12 Fab complex wasdetermined.

8D8 Fab and 5F12 Fab animo acid sequences are shown below, with VH andVL sequences bolded and CDR regions bolded and underlined.

8D8 Fab Hc: (SEQ ID NO: 1828) QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGM IWGFGSTDYNSALKS RLSITKDNSKSQFFLKMNSLQTDDTAMYYCARIHTTAGSY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVDG 8D8 Fab Lc: (SEQ ID NO: 1829)DIVMTQSQKFMSTSIGDRVSVTCKASQNVGTNVA WYQQKPGQSPKALVYS TSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQYNSYPLT FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 5F12Fab Hc: (SEQ ID NO: 1830) QVQLKQSGTELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIAR IYPGNGNTYHNEKFKG KATLTAEKSSSTAYMQLSSLTSEDSAVYFCAREGLYYDYDRYFDY WGQGTALTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDEVDG 5F12 Fab Lc: (SEQ ID NO: 1831)NIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMH VVYQQKPGQPPK LLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDP PAFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

FIG. 59 illustrates an aspect of a 3P10 Fab::GFRAL::25M22 Fab complex bya ribbon diagram. The Fab fragments interact with an asymmetric unit ofGFRAL in the 3P10 Fab::GFRAL::25M22 Fab complex crystal. The crystalstructure also showed that GFRAL epitope residues 290-312 were presentedto the 3P10 antibody and GFRAL N-terminal epitope residues 130-157 werepresented to the 25M22 antibody heavy chain CDR regions

FIG. 60 illustrates 8D8 Fab and 5F12 Fab binding sites on the GFRALprotein. Residues on the GFRAL protein that are important for Fabbindings are shown as stick models.

An analysis of the GFRAL epitope presented to 8D8 Fab in the 8D8Fab::GFRAL::5F12 Fab complex crystal structure showed that 8D8'smechanism of action is that of a competitive GDF15 inhibitor andinvolves blocking GDF15 binding to GFRAL. Exemplary core interactioninterface amino acids on the GFRAL protein and on the 8D8 Fab CDRs forthe heavy and light chains are shown in FIG. 61.

FIGS. 62A, 62B, 62C and 62D illustrate the core and boundary amino acidresidues in the GFRAL/8D8 Fab interaction interface. FIG. 62Aillustrates the structure of GFRAL with the core 8D8 interactioninterface amino acids (GFRAL epitope residues) on GFRAL highlighted in aspace-filled surface model. FIG. 62B also illustrates the core interfaceamino acids on GFRAL covered by 8D8 in a space-filled surface model.FIG. 62C illustrates the structure of GFRAL in a ribbon diagram withboundary 8D8 Fab interaction interface amino acids on GFRAL (GFRALepitope residues) highlighted in a space-filled surface model. FIG. 62Dalso illustrates the boundary interface amino acids on GFRAL covered by8D8 in a space-filled surface model.

FIGS. 63A, 63B, 63C and 63D illustrate the core and boundary amino acidresidues in the GFRAL/5F12 Fab interaction interface. FIG. 63Aillustrates the structure of GFRAL with the core 5F12 interactioninterface amino acids (GFRAL epitope residues) on GFRAL highlighted in aspace-filled surface model. FIG. 63B also illustrates the core interfaceamino acids on GFRAL covered by 5F12 in a space-filled surface model.FIG. 63C illustrates the structure of GFRAL in a ribbon diagram withboundary 5F12 Fab interaction interface amino acids on GFRAL (GFRALepitope residues) highlighted in a space-filled surface model. FIG. 63Dalso illustrates the boundary interface amino acids on GFRAL covered by5F12 in a space-filled surface model.

GFRAL amino acids at the interface of the GFRAL/8D8 Fab complex areshown in Table 47. To compare the interaction interface amino acids inGFRAL/8D8 Fab and GFRAL/GDF15 complexes, Table 47 further lists theGFRAL amino acids at the interface of the GFRAL/GDF15 complex, which isalso shown in Table 38. Table 47 illustrates that core interactioninterface amino acid residues on GFRAL that bind to GDF15 overlaps withthe core interaction interface amino acids in the GFRAL/8D8 Fabinteraction.

The amino acid sequence of a full-length precursor human GFRAL proteinis shown below (see also Example 12, part C):

GFRAL sequences SEQ ID NO: 1797        10         20         30         40 MIVFIFLAMG LSLENEYTSQTNNCTYLREQ CLRDANGCKH         50         60         70         80AWRVMEDACN DSDPGDPCKM RNSSYCNLSI QYLVESNFQF        90        100        110        120 KECLCTDDFY CTVNKLLGKKCINKSDNVKE DKFKWNLTTR        130        140        150        160SHHGFKGMWS CLEVAEACVG DVVCNAQLAS YLKACSANGN       170        180        190        200 PCDLKQCQAA IRFFYQNIPFNIAQMLAFCD CAQSDIPCQQ        210        220        230        240SKEALHSKTC AVNMVPPPTC LSVIRSCQND ELCRRHYRTF       250        260        270        280 QSKCWQRVTR KCHEDENCISTLSKQDLTCS GSDDCKAAYI        290        300        310        320DILGTVLQVQ CTCRTITQSE ESLCKIFQHM LHRKSCFNYP       330        340        350        360 TLSNVKGMAL YTRKHANKITLTGFHSPFNG EVIYAAMCMT        370        380        390 VTCGILLLVMVKLRTSRISS KARDPSSIQI PGEL

TABLE 47 residues on GFRAL Residues on GFRAL that bind to that bind toGDF15 8D8 in in GFRAL/8D8 Fab structure Core interaction Coreinteraction Boundary interaction interface amino acids interface aminoacids interface amino acids LEU132 Glu136 LEU132 ALA135 Ala137 GLU133GLU136 Val139 VAL134 VAL139 Gly140 ALA135 GLY140 Asp141 GLU136 VAL142Val142 ALA137 ASN145 Val143 CYS138 ALA146 Cys144 VAL139 LEU148 Asn145GLY140 ALA149 Ala146 ASP141 LEU152 Gln147 VAL142 LYS153 Phe173 VAL143ILE196 Asn177 CYS144 PRO197 Ile178 ASN145 GLN200 Pro179 ALA146 SER201Asn181 GLN147 ALA204 Ile182 LEU148 LEU205 Met185 ALA149 SER150 TYR151PHE174 TYR175 ALA169 ALA170 ILE171 ARG172 PHE173 GLN176 ASN177 ILE178PRO179 PHE180 ASN181 ILE182 ALA183 GLN184 MET185 LEU186 ALA187 PHE188CYS189 Core interaction interface amino acid residues on GFRAL that bindto both GDF15 and 8D8 Fab are shown in bold.

An analysis of the GFRAL epitope presented to 5F12 Fab in the 5F12Fab::GFRAL::8D8 Fab complex crystal structure showed that 5F12'smechanism of action is that of a non-competitive GDF15 inhibitor anddoes not involve blocking GDF15-binding to GFRAL. Exemplary coreinteraction interface amino acids on the GFRAL protein and on the 5F12Fab CDRs for the heavy and light chains are shown in FIG. 64.

GFRAL amino acids at the interface of the GFRAL/5F12 Fab complex areshown in Table 48. To compare the interaction interface amino acids inGFRAL/5F12 Fab and GFRAL/RET complexes, Table 48 further lists the GFRALamino acids at the interface of the GFRAL/RET complex, which is alsoshown in Table 40A. Table 48 shows core interaction interface amino acidresidues on GFRAL that bind to both RET and 5F12 Fab. Residues on GFRALwhich are critical for GFRAL/5F12 interactions: 5F12 binding epitope onGFRAL reveals a non-competitive inhibitory mechanism of action ofblocking GFRAL/RET interactions.

TABLE 48 residues on GFRAL that bind to RET in RET/GFRAL/GDF15 Modelresidues on GFRAL that bind to Core interaction 5F12 in 5F12/GFRALstructure interface amino acids Core interaction Boundary interaction onGFRAL interface amino acids interface amino acids Gln246 Arg234 CYS233Arg247 Arg238 ARG234 Arg250 GLN241 ARG235 Lys251 Ser242 HIS236 Cys252Lys243 TYR237 Asp255 Trp245 ARG238 Glu256 Gln246 THR239 Asn257 Thr249PHE240 Cys258 Arg250 GLN241 Ile259 Lys251 SER242 Ser260 Cys252 LYS243Thr261 His253 CYS244 Leu262 Asp255 TRP245 Thr297 Asn257 GLN246 Gln298Cys258 ARG247 Ser299 Ser260 VAL248 Thr261 THR249 Leu262 ARG250 LYS251CYS252 HIS253 GLU254 ASP255 GLU256 ASN257 CYS258 ILE259 SER260 THR261LEU262 SER263 LYS264 ASP266 LEU267 THR268 SER272 ASP274 CYS275 ALA278CYS269 SER270 SER302 LEU303 ILE306 HIS309 LEU311 MET310 SER315 CYS316Core interaction interface amino acid residues on GFRAL that bind toboth RET and 5F12 Fab are shown in bold.

The contents of all references described herein are hereby incorporatedby reference.

Other embodiments are within the following claims.

SEQUENCE LISTING

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing. The CRF entitled13370-090-888_SEQ_LISTING.txt, which was created on Aug. 23, 2018, andis 968,136 bytes in size, is incorporated herein by reference in itsentirety.

Lengthy table referenced here US20190092866A1-20190328-T00001 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20190092866A1-20190328-T00002 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20190092866A1-20190328-T00003 Pleaserefer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190092866A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1.-106. (canceled)
 107. A method of treating cachexia in a subject,wherein the method comprises administering to the subject an antibodythat binds GDNF Family Receptor Alpha-like protein (GFRAL), wherein theantibody comprises a heavy chain CDR1 comprising SEQ ID NO:46, a heavychain CDR2 comprising SEQ ID NO:137, a heavy chain CDR3 comprising SEQID NO:225; a light chain CDR1 comprising SEQ ID NO:301, a light chainCDR2 comprising SEQ ID NO:376, and a light chain CDR3 comprising SEQ IDNO:426.
 108. The method of claim 107, wherein the antibody comprises aheavy chain variable region having at least 90% identity to SEQ IDNO:1982 and a light chain variable region having at least 90% amino acididentity to SEQ ID NO:1997.
 109. The method of claim 107, wherein theantibody comprises a heavy chain variable region having at least 95%identity to SEQ ID NO:1982 and a light chain variable region having atleast 95% amino acid identity to SEQ ID NO:1997.
 110. The method ofclaim 107, wherein the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:1982 and a light chain variable region comprisingSEQ ID NO:1997.
 111. The method of claim 107, wherein the antibody is amonoclonal antibody.
 112. The method of claim 107, wherein the antibodyis a humanized antibody.
 113. The method of claim 107, wherein theantibody is an IgG1, an IgG2, or an IgG4 antibody.
 114. The method ofclaim 110, wherein the antibody is an IgG1, an IgG2, or an IgG4antibody.
 115. The method of claim 107, wherein the subject has achronic disease selected from the group consisting of liver cirrhosis,hyperthyroidism, Parkinson's disease, cancer, chronic renal disease,chronic obstructive pulmonary disease, AIDS, tuberculosis, chronicinflammatory disease, sepsis, muscle wasting, and anorexia nervosa. 116.The method of claim 107, wherein the subject has cancer.
 117. The methodof claim 116, wherein the subject has elevated levels of GDF15.
 118. Themethod of claim 107, wherein the subject is undergoing treatment forcancer.
 119. The method of claim 107, wherein the subject has elevatedlevels of GDF15.
 120. The method of claim 110, wherein the subject has achronic disease selected from the group consisting of liver cirrhosis,hyperthyroidism, Parkinson's disease, cancer, chronic renal disease,chronic obstructive pulmonary disease, AIDS, tuberculosis, chronicinflammatory disease, sepsis, muscle wasting, and anorexia nervosa. 121.The method of claim 110, wherein the subject has cancer.
 122. The methodof claim 121, wherein the subject has elevated levels of GDF15.
 123. Themethod of claim 110, wherein the subject is undergoing treatment forcancer.
 124. The method of claim 110, wherein the subject has elevatedlevels of GDF15.
 125. The method of claim 107, wherein the methodcomprises administration of at least one additional therapeutic agent.126. The method of claim 110, wherein the method comprisesadministration of at least one additional therapeutic agent.
 127. Themethod of claim 107, wherein the antibody is formulated as apharmaceutical composition further comprising a pharmaceuticallyacceptable carrier.
 128. The method of claim 110, wherein the antibodyis formulated as a pharmaceutical composition further comprising apharmaceutically acceptable carrier.
 129. A method of treatinginvoluntary weight loss in a subject, wherein the method comprisesadministering to the subject an antibody that binds GFRAL, wherein theantibody comprises a heavy chain CDR1 comprising SEQ ID NO:46, a heavychain CDR2 comprising SEQ ID NO:137, a heavy chain CDR3 comprising SEQID NO:225; a light chain CDR1 comprising SEQ ID NO:301, a light chainCDR2 comprising SEQ ID NO:376, and a light chain CDR3 comprising SEQ IDNO:426.
 130. A method of treating a cancer in a subject having elevatedlevels of GDF15, wherein the method comprises administering to thesubject an antibody that binds GFRAL, wherein the antibody comprises aheavy chain CDR1 comprising SEQ ID NO:46, a heavy chain CDR2 comprisingSEQ ID NO:137, a heavy chain CDR3 comprising SEQ ID NO:225; a lightchain CDR1 comprising SEQ ID NO:301, a light chain CDR2 comprising SEQID NO:376, and a light chain CDR3 comprising SEQ ID NO:426.